There is a growing trend in the pharmaceutical industry towards accelerated development of therapeutics to address unmet medical needs. The shorter timelines coupled with increasing complexity of the development candidates and/or delivery systems presents extraordinary challenges for managing the chemistry, manufacturing and controls activities. Key to regulatory and operational success is a risk based approach to management of manufacturing, product quality and supply chain. This workshop will address important concepts for balancing and mitigating a variety of risks (safety, economic or regulatory, etc.) towards successful clinical development, regulatory approval and commercialization of therapeutic oligonucleotides.

Characterizing large peptide impurities with confidence is a challenge. Affordable High Resolution Mass Spectrometry (HRMS) systems, coupled to UHPLC open new opportunities. This presentation will show through real case studies, how in-house HRMS technology allows fast identification and enables chemists to build up knowledge for better manufacturing processes.

Chemo-enzymatic peptide synthesis (CEPS) technologies present the opportunity to manufacture long peptides with very significantly increased yields. This has been demonstrated at benchtop scale for the 39-mer peptide exenatide using a 21-mer peptide ester and an 18-mer peptide, where the "peptiligase" coupling efficiency exceeded 97% and the final overall yield exceeded 50%. In particular, the fragment strategy, choise or C-terminal ester, enzyme development and reaction design at gram scale will be elaborated.

Recent advances towards mRNA therapeutics prompt a need for robust analytical methods capable of characterizing this new class of drugs. We demonstrate the use of specific enzymatic digestions followed by LC/MS mass fingerprinting to evaluate the critical quality attributes (CQAs) of mRNA therapeutics.

Messenger RNA (mRNA) therapeutics are an exciting new modality for genome editing, gene replacement, antibody expression and vaccines. A broad overview of mRNA applications, design, manufacturing, quality control and GMP production will be provided.

Chemo-enzymatic peptide synthesis (CEPS) has recently emerged as a powerful tool for the synthesis of a wide range of peptides and proteins.1 Using Exenatide as a model substrate we report an industrial assessment of various aspects of the CEPS technology from the standpoint of large scale cGMP production of peptide based drugs.

Subcutaneous injection is the most common administration route for peptide therapeutics. Catabolism at the injection site can be a major degradation pathway for many peptides, and in some cases it can significantly affect pharmacokinetics, particularly bioavailability. The presentation will describe the development and the application of a liquid chromatography-high resolution mass spectrometry-based in vitro assay to study the metabolic stability in the subcutaneous tissue of peptides in human and preclinical species (rat and minipig). The assay allows for the simultaneous determination of degradation rate (half-life) of the parent peptide and identification of metabolites generated from enzymatic proteolysis. Validation was first performed using known therapeutic peptides such as human insulin, GLP-1 and LHRH analogs. Finally, the method was successfully applied in a real peptide drug discovery scenario, showing a good correlation between in vitro metabolic stability and in vivo bioavailability after SC administration in rats, but also between in vitro and in vivo metabolism. The implementation of this assay in the screening cascade can be beneficial in many ways, including the possibility to drive the synthesis of peptides with higher bioavailability and prioritize compounds for in vivo studies, thus reducing the number of in vivo procedures.

SB3000 are working with a disruptive technology for continuous manufacturing of peptides. The benefits are many; notably no need for scale up processes, a very predictable and transparent manufacturing process leading to pristine data on both purity and yield. This address both the FDA’s push for more predictable manufacturing processes, and environmental and cost issues facing the manufacturing society.

During this workshop we will present the broad and diverse pipeline of ProQR Therapeutics and the focus will be on the planning and manufacturing of drug substance supply for the different (pre-)clinical programs within ProQR. Moreover, we will discuss the challenges associated with having very different DS supply needs in combination with very short and strict timelines.

At Avecia, mass spectrometers of varying capabilities are used in variety of oligonucleotide characterization studies. These include molecular weight determinations, sequence confirmations, and impurities characterization. Different approaches along with appropriate examples will be presented; suitability of different mass spectrometers for each characterization will also be discussed.

MS coupled to the HPLC-purity-method is used within a comprehensive control strategy to ensure product quality. Therefore, MS is rapidly developing into a routine QC technology. With our approach we address this challenge by an efficient LC-UV-MS peak purity workflow allowing to fully exploit the capabilities of state-of-the-art MS analysis.

The SPPS technology is now more than 50 years old, and it is acknowledged as the most versatile standard approach for making peptides. Still, the biphasic system, where the product-being-made is attached to the solid phase, presents a number of challenges for developing and executing highly efficient processes in a complex environment. To overcome these challenges, there is a need for an increased level of process and chemistry understanding and control of the actual systems. By taking a look at the processes with a renewed combination of theoretical and practical tools, the first steps in generating further understanding are taken. In this presentation concepts and data are shared.

Synthetic oligonucleotides hold great potential to serve as therapeutics for the treatment of human diseases. In this presentation, we illustrate how different mass spectrometry methodologies can be used to characterize these complex molecules.

This talk will include an introduction to using CRISPR, starting with the repurposing of the bacterial adaptive immune system, CRISPR's use as a research tool, and therapeutic uses. This overview will include different Cas9 system, choice of target sites, high-throughput testing, preclinical assays, an overview of bioinformatics and the path to therapeutic use.

FDA and EU National Authorities have similar approaches to review and approval of clinical trials, however key areas of difference need to be understood to achieve successful applications. This presentation provides advice for oligonucleotide companies covering: 1) Pre-submission dialogue 2) Differences in application content and Regulatory Agency review approaches and 3) Commitments during and after trial completion.

We will discuss recent advancements at Editas, developing technologies that drive reagent quality and enable evaluation of safe and effective CRISPR-based medicines. These include aspects of our methods and quality control processes for guide RNA and ribonucleoprotein complexation, as well as novel guide RNA chemistries.

Deamidation and aggregation were observed during the chemical synthesis and during the release of a peptide from the formulated product. This presentation will focus on the assessment of different peptide variants designed to mitigate these risks and allow the nomination of a more stable peptide for clinical development.

Mammalian genomes contain tens of thousands of sites where DNA double-strand breaks (DSBs) occur, many recurrently. We find that a significant number of nominal Cas9 off-target sites are in fact background DSBs, suggesting that background DSBs can inflate measures of Cas9 imprecision. Allowing for this, we find ~45% of guides under our standard conditions have no detected off-target activity.

The biggest RP silica based HPLC process is doomed by the strong tendency of insulin and its analogues to aggregate on the column. CIP (Cleaning in Place) by aggressive solvents is necessary to balance process economy. The development process of better separation tools (stationary phases) will be discussed using real industrial examples.

To fulfill a need for non-viral delivery methods intended for ex vivo cell therapy and in vivo local administration, we developed a peptide-based technology that enables efficient entry of CRISPR ribonucleoprotein complex in cells. These amphiphilic cationic peptides are optimized to deliver proteins in hard-to-transfect cells, including Natural Killer and airway epithelia cells.

Corden Pharma Colorado has an impressive track record in peptide manufacturing innovation. Applications and technologies from our peptide learnings are considered for oligonucleotide manufacturing processes. The traditional oligonucleotide production concepts are challenged from an engineer's perspective and new approaches to manufacturing will be presented.

Decades after the first clinical testing of oligotherapeutic drugs, there exists a sparsity of specific regulatory guidances to aid in the appropriate design of preclinical programs for this class of molecules. This presentation will summarize key considerations for the successful preclinical development of oligonucleotide drugs, including selection of appropriate animal models, test article controls, dose scaling, pharmacokinetic determinations, and special study design considerations.

Peptides and Oligonucleotides often use a manufacturing process train of SPPS for synthesis, a deprotection step, purification by chromatography and isolation by lyophilization. Alternative  or telescoped processes that have been successfully used for some peptides.  Examples or case studies for these will be presented.  In addition, some hypothetical concepts will be introduced that could also transform processes for both “Tides”.

Delivery is a key challenge in developing CRISPR based therapeutics. In this presentation, I will present polymer based delivery systems for CRISPR/Cas ribonucleoprotein. In vivo proof-of-concept data in muscle tissue and other tissues will be presented.

We present the first study in pharmaceutical sciences that the structure of insoluble aggregate of a representative peptide drug has been determined at a molecular level. This enables us to identify residues that lead to backbone rearrangement, which can facilitate optimization of peptide drugs with lower risks of aggregation.