Arcturus has developed a novel platform called LUNAR®, a proprietary lipid-enabled delivery system for RNA medicines. The challenge for mRNA therapeutics, however, is delivering the mRNA to the target cell type. Arcturus’ LUNAR platform technology provides effective delivery of mRNA to appropriate cell types in liver, lung and other tissues.

Although, the concept of the “solid-phase” by itself is totally green since the point that all reactions are carried in only one reactor with a clear minimization of mechanical losses and of mass transfer, SPPS is using highly reprotoxic solvents, being classified as a Substance of Very High Concern.  Our work is a first and important step toward a greener SPPS in terms of replacement of toxic solvents, waste generated, and energy efficiency.

The discussion will focus on all aspects of drug product development. We will start with analytical methods required for drug product, including in process controls, QC release methods, and stability, and how they differ from those for drug substance. In addition, we will discuss preformulation and formulation development of peptides to support non-clinical research studies, toxicity studies, and clinical programs. A third topic area to cover will be the selection, design, and development of delivery systems for peptides.

Anionic carriers represent a safe way for delivering of nucleic acids due to much reduced immunostimulation. Anionic carriers therefore open new routes for repeated dosing or targets residing within antigen presenting cells.

Enzymes have been widely applied in manufacturing, where they are valued for their high efficiency. When compared to traditional synthetic chemistry, processes using enzymes offer reductions in energy costs and hazardous waste. However, the applications of existing enzymes are severely limited by the scope of the natural chemical reactions they can catalyze. Using directed evolution—the subject of the 2018 Nobel Prize in Chemistry—it is possible to merge the environmental benefits of enzymes with the versatility of chemical manufacturing to provide a sustainable route to useful products. We have used directed evolution to create an evolved variant of tryptophan synthase that is able to produce nearly 100 noncanonical amino acids in a single step from cheap materials, with water as the only byproduct. This method can be broadly used to manufacture noncanonical amino acids on gram to kilogram scales, and the enzyme can be easily separated, allowing it to be reused or biodegraded. Unlike existing methods for producing ncAAs, our approach does not require hazardous chemicals such as cyanide or rare metals such as palladium. As enzyme engineering continues to expand scope of available products, this may lead to a new, more efficient path to manufacturing noncanonical amino acids.

Peptides bridge small molecules and biologics, not only in their size, but also in specificity and selectivity. These unique properties of peptides require specialized consideration when designing and executing the safety studies to support first-in-human clinical trials. This talk will outline and discuss the pharmacokinetics and bioanalysis of peptides and the design of toxicology studies for peptides.

We will describe structure‐guided, rational optimization of nanoparticle carriers for delivery of long RNAs to achieve mRNA-mediated protein replacement and CRISPR/Cas gene editing. For example, we will show how mDLNPs effectively delivered fumarylacetoacetate hydrolase (FAH) mRNA that normalized liver function and significantly extended survival in difficult‐to‐treat Hepatorenal Tyrosinemia Type I (HT‐1) mouse model.

The talk will discuss development of an enhanced and flexible control strategy to support antisense oligonucleotides programs at Biogen. By leveraging knowledge accumulated from prior experience, the control strategy uses risk-based scientific approach to determine control points and testing scheme to reduce redundancies, increase quality and responsiveness, show process control, and enable rapid release of material.

Translate Bio has demonstrated the potential of messenger RNA (mRNA) therapeutics as a new approach for the treatment of diseases caused by protein or gene dysfunction. We have observed successful production of desired proteins through delivery of lipid nanoparticle-encapsulated mRNA to various target tissues. Our proprietary lung delivery system has supported the advancement of our lead program in cystic fibrosis into the clinic. We have applied this delivery technology via other routes of administration in multiple target tissues to assess the potential across a broad array of diseases. We present here various routes of administration of mRNA in lipid nanoparticles which result in successful protein production and distribution in multiple target tissues.

Hybrid SPPS/LPPS is a well known method of peptide synthesis. A key benefit is that coupling peptide  fragments insolution (LPPS) can significantly reduce solvent use, resulting in greener synthetic approaches.  For synthesis of small fragments (< 10 mer) SPPS provides certain advantages over full solution phase chemistry approaches peptides  can be synthesized more rapidly and isolated with a higher purity.  These small peptide fragments can be well characterized with high purity which make them ideal candidates as regulatory starting materials.  As a peptide chain grows longer in SPPS challenges with increased levels of deletion  impurities can lead to marked increases in costs associated with purification, largely due to decreasing coupling efficiency related to mass transfer resistances and steric hindrance.   Additionally, convergent approaches to peptide synthesis are far more amenable to alternative reactor designs such as flow infrastructure given the low concentration, high potency and high value of the peptide assets.  Control strategies for convergent approaches can also be significantly simplified given that peptides are routinely terminally purified through chromatographic means.  Synthesis impurities that are markedly different in molecular weight which are expected by hybrid SPPSLPPS /are often more effectively separated in preparative chromatographic systems due to large differences in retention time.  Despite the potential large upsides of hybrid SPPS/LPPS strategies, most companies manufacturing peptides presently do not utilize this strategy. The focus of the presentation will be on efforts at Eli Lilly to develop novel flow methodology to produce commercial peptides in internal or external facilities. This methodology has been applied extensively in small molecule manufacturing and is known to improve yield, purity and can reduce solvent usage by up to 50%. Our initial results indicate that this approach could be transformative to the peptide industry.    In addition, the presentation will deliver a scientific and business  case for establishing short to medium size peptide fragments (<10 mer ) as regulatory starting materials.

This presentation will focus on the FDA requirements but also highlight the roles of the sponsor and the manufacturer. Understanding the manufacturing process, developing appropriate analytical methods allowing for reliable monitoring on the impurities and setting the product specifications are key elements to address any potential safety concerns. The IND dossier should address all of the sections related to the process, analytical methods and justification of the specifications. An overview of the various sections will be presented.

At Genevant, we are pursuing a modality agnostic “pan-RNA” approach to develop a diverse pipeline of RNA therapeutics. Genevant’s lipid nanoparticle (LNP) platform has enabled several clinical trials for nucleic acid therapeutics and is used in the first approved RNAi product. It has been used to deliver RNAi payloads targeting both viral and endogenous gene targets, and mRNA in vaccine applications. In this presentation, we will describe further application to mRNA payloads, showing how structural changes to component lipids can influence biological performance in rodents and NHP. We have also developed a potent ligand conjugate platform for delivery of oligonucleotides to the liver, similarly deploying it against both viral and endogenous gene targets. We describe its further refinement, resulting in a substantial increase in activity paired with a more rapid onset. The potential of these platforms for extrahepatic delivery will also be discussed.

As a part of successful development program for a peptide product, a careful planning on the regulatory strategy is necessary. It is critical to have a good understanding on the phase-dependent regulatory requirements based on the unique characteristics of the peptides to avoid pitfalls and delay. The regulatory issues may be related to the design of pre-clinical studies, impurities determination and qualification, bioassay, immunogenicity, and comparability following the manufacturing changes. Any of these issues can present significant regulatory challenges for the development program. The presentation will discuss how to navigate these regulatory issues during R&D and different phases of clinical investigation.

The talk will cover issues related to delivery of mRNA products from discovery through development for different therapeutic areas and routes of administration – from local delivery of prophylactic and therapeutic vaccines to systemically delivered therapeutics.

As more and more oligonucleotides are entering preclinical and clinical evaluation, both analytical and bioanalytical techniques play an ever more important role. This workshop will explore the most commonly used analytical and bioanalytical techniques and their applications throughout the oligonucleotide development process. You will learn about the challenges, considerations, perspectives and expectations for both analytical studies and bioanalysis in oligonucleotide drug development and manufacturing. You will also hear a variety of case studies from industry experts to illustrate lessons learned.

The presentation will provide an overview of gelation and aggregation in formulations and methods to prevent or delay formulation instability. It will also provide an overview of injection site precipitation and the consequences, as well as methods to prevent or delay precipitation.

This workshop will provide an overview of CMC requirements for peptides including discussions on a wide variety of topics including 1) Risk assessments 2) Genotoxic impurities and justification 3) Related substance impurities and qualification justification 4) Report results vs a defined specification 5) Compendial Tests – Ph Eur vs USP vs Harmonized or not harmonized 6) Regulatory starting materials 7) Method Validation requirements at Phase 2- US vs EU and more.

Peptides are typically formulated in aqueous-based formulations and aggregation and fibrillation can pose significant challenges. Formulation in biocompatible non-aqueous systems can often provide greater control over aggregation/fibrillation whilst providing a means for safe and effective injections. This presentation will focus on mechanisms of solvent influence on aggregation and review several case studies.

Many synthetic peptides are currently worldwide approved as drugs. Nevertheless, identification of critical quality attributes (CQAs), analytical challenges and characterization issues are still discussed in the community. This presentation will also focus on regulatory starting materials and related substance impurities qualification.

Injectable drugs that are insoluble at physiological pH are formulated at a different pH or containing non-physiological concentrations of salts and excipients to enhance the solubility of the drug substance.   Such formulations upon injection have a tendency to precipitate at the injection site causing injection site reaction. Here we present in vitro and in vivo screening methods to develop formulations to prevent injection site precipitation.

This presentation will discuss higher-order structure and hydrogel assembly of oligopeptides, characterization of the rheological behaviour, factors effecting gel properties and biomedical applications of peptide with gelation characteristic.

The FDA recently released a new draft guidance defining the equivalence of a rDNA peptide product and a synthetic peptide product. The draft guidance enables generic manufacturers of peptide drugs to file an Abbreviated New Drug Application (ANDA) for synthetic peptide drug products that refer to listed drugs of rDNA origin. Since the processes for manufacturing the generic and reference drug (RLD) are not equivalent, peptide drugs can be associated with impurities. Impurities can result from changes in the sequences due to deletions, insertions, integration of incorrect amino-acids and modifications and also impurities related to the synthetic production. The FDA draft guidance requires manufacturers to prove that the synthetic peptide product does not contain impurities that have an increased affinity for major histocompatibility complexes and potential for engaging immune response, which may drive undesired anti-drug antibody development. We have used both immunoinformatics-driven analysis and in vitro validation assays to perform immunogenicity risk assessment of peptide generics. This combination of in silico and in vitro tools is referred to the PANDA assay which can be used to support generic peptide drug equivalency in an ANDA application. This presentation will provide insight as to the process of performing the PANDA assay, illustrating the process with two case studies (such as Calcitonin and Teriparatide).

Sebastien Burel, Ph.D., Executive Director, Nonclinical Development, Ionis Pharmaceuticals (Invited)

The stability of therapeutic proteins can be influenced by multiple factors leading to chemical and physical instabilities. This case study of a synthetic peptide addresses the key development challenges during formulation development. A narrow pH-window, gel formation at higher concentration and incompatibility with silicone oil and preservatives made it challenging to develop a multi-use formulation for parenteral use. The different siliconization processes of glass barrels and packaging material (baked versus spray-on) had a great impact on protein aggregation. In this study, the siliconization of plunger stoppers could be identified as one trigger factor for aggregation. The implementation of silicone-free packaging material and optimization of the formulation led to a stable parenteral formulation. Performing compatibility testing of different packaging materials and investigating the interaction of preservatives with peptides early during formulation development could be beneficial with respect to shortened clinical timelines.

The establishment of the UHPLC technology opened the door to efficient and robust separation of many impurities in synthetic peptides. Nevertheless, the separation of some impurities remains challenging even using most recent UHPLC RP techniques and development approaches. Among these “separation resistant” impurities are the D-His1 analogues of many GLP peptides. Up to now special methods such as IEX-HPLC or chiral GC amino acid analysis must be applied in these cases.

An alternative method for the quantitative determination of D-His1-Glucagon in Glucagon will be presented as case study. The method comprises a proteolytic digest of Glucagon followed by RP UHPLC quantitation of the histidine-containing peptide fragment. The method can be validated for routine analysis and represents an example for a general application of the approach for peptides with different kinds of impurities that were difficult to separate otherwise.

Charge heterogeneity can occur in synthetic peptides due to chemical degradation (e.g. oxidation, deamination, etc.) during synthesis, purification and storage which can impact safety and efficacy. In this study, we explored the feasibility of using capillary isoelectric focusing to characterize the charge heterogeneity of >30 amino acids peptides.

DCR-PHXC, an siRNA targeting LDHA, is being developed for the treatment of primary hyperoxaluria and has demonstrated proof of concept in a Phase I study.  This case study will be used to examine CMC and nonclinical strategies that may be helpful for early stage oligonucleotide development programs.

Physical and chemical instability of peptides pose a significant risk to the successful development of a solution formulation product. While methods for predicting chemical stability have been developed, the prediction of physical stability remains a key gap in industry. Accelerated approaches to screen formulations are developed for rank-ordering; however, the extent to which the rank-order remains at relevant storage conditions has yet to be established. Therefore, current formulation selection requires robust long-term data at relevant storage conditions, which is lengthy and demands significant resources. The peptide discussed herein has a narrow pH window for optimal physical and chemical stability. Indeed, fibril formation has been observed when pH < 8 however above this pH significant chemical degradation occurs. As the impact of excipients required for formulation stability and sterility are difficult to determine a priori, it becomes necessary to monitor large screens of unique candidate formulations for analysis of physical and chemical stability. In an ideal situation, prediction studies would be performed on freshly prepared peptide or utilize short incubation times for formulations of interest. In this work, the fluorescence-based assay implemented to study physical stability involves the simultaneous heating and agitation of peptide formulations to accelerate peptide fibril formation. Solutions of peptide in the absence of excipients display Arrhenius behavior, where increased temperatures lead to faster fibril formation. Surprisingly, the addition of stabilizing excipients essentially reverses this trend: fibril formation is observed at lower temperatures but is inhibited at higher temperatures. Of greater importance, the data collected at lower temperatures better reflects the behavior of peptide formulations under the relevant real-time storage conditions. These results are supported by isothermal titration calorimetry studies which report the enhanced binding of stabilizing excipients to the peptide at elevated temperatures. Further NMR experiments provide molecular-level insight into this phenomenon. These finding can have great impact on product development strategy as we gain further insight on peptide-excipient interactions. The growing understanding and increased ability to predict peptide physical stability will enable a more robust and efficient development of peptide therapeutics.

Peptide/protein biotherapeutics fall in a grey area between biologics and small molecules when it comes to assessment of genotoxic impurities. Although, the ICH M7 guideline considers biologics out of scope, some of the concepts and strategies described there can be applied to all process-related impurities. Nonetheless, there are unique properties of biologics that must be taken into consideration when performing a genotoxicity assessment. This talk will describe the output of a Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee Workgroup that formed to address these topics.