by Catarina Carrao
This piece represents the views of the author and not necessarily the views of Informa Connect.
Peptides represent a unique class of pharmaceutical compounds, molecularly poised between small molecules and proteins, yet biochemically and therapeutically different from both. With a fundamental signaling action for many physiological functions, they present an opportunity for therapeutic intervention that closely mimics natural pathways1. There are a variety of challenges for the development of peptide therapeutics: (i) their intrinsic complexity for manufacturing, characterization and formulation, which increases their costs; (ii) the preparation of peptides for other than injection is still a challenge, and requires extensive development; (iii) expectations from the agencies for the control of the purity profile of peptidic Active Pharmaceutical Ingredients (API) are becoming increasingly complex and require a thorough understanding of the nature and the fate of impurities2.
To address the first concern, many peptides are developed for niche markets with smaller drug product demands; where the typical high potency of peptides, offsets the costs of the API2.
Secondly, efforts are underway to improve the oral availability of peptide therapeutics by increasing drug stability in the gastrointestinal tract; formulating peptides with permeability enhancers; and, improving the peptide Central Nervous System (CNS) availability through conjugation to carrier molecules, or delivery in nanoparticles1. Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and pharmaceutical peptide safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance3. For example, manufacturing of peptides on solid support does not rely on intermediates, which can be crystallized to remove impurities in the course of the synthesis; but rather, progress through the so-called telescope steps, with final purification only2. Therefore regulators expect a high level of process understanding and control; and, specify in the regulations that starting materials (e.g. for protected aminoacid derivatives) should address impurities based on the origin, fate and purge; and, use validated analytical methods2.
A recent study based on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) establishes new nuclear magnetic resonance (NMR) peptide sequencing methodology, that overcomes key limitations of basic methods in identifying small structural changes or minor impurities that might affect efficiency and safety of therapeutic peptides3. HiFSA sequencing produces simultaneously definitive identity and purity information, allowing for API quality assurance and control (QA/QC); and achieving full peptide analysis via NMR building blocks, in a process that serves both research and commercial applications3. One can expect a significant increase in interest in peptide pharmaceuticals, primarily due to their low toxicity and wide range of possible molecular targets4. Improvements in peptide screening and computational biology will continue to support peptide drug discovery1.
Oligonucleotides (oligos) have been under clinical development for almost the past 30 years, beginning with antisense oligonucleotides (ASOs) and aptamers; and, followed about 15 years ago by silencing RNAs (siRNAs)5. Oligonucleotides, as peptides, resemble biologics in some ways, because of their molecular complexity, but are much smaller in size; leading to unique concerns in the design of control strategies for these types of molecules. A good starting point to designing therapeutics in this class, is the usage of natural peptides to improve physical and chemical properties such as stability and bioavailability; and, in relation to methods of manufacture, chemical synthesis, recombinant DNA technology, or extraction from natural sources, continue to be the preferred methodology currently in use6.
Despite considerable progress, two major obstacles stand in the way of widespread application of oligonucleotide therapeutics and regulatory approval: (i) drug safety, and (ii) delivery. The administration of oligonucleotides has been associated with the activation of innate immunity through interactions with toll-like receptors (TLRs); with some oligonucleotides binding to TLRs, and inducing immune responses similar to those induced by viral and bacterial RNA and DNA7. Different sequence motifs have been identified as agonists of TLR family members; as such, avoiding these sequence motifs and using chemical modifications can minimize these immune-stimulatory effects8.
The use of some siRNA therapeutics in clinical trials may be associated with another liability: inflammatory responses to the lipid nanoparticle formulations used to promote the uptake of siRNAs7. Lipid nanoparticles are known to induce a complex antiviral-like response of innate immunity9. To diminish the immune-stimulatory effects of the formulations, siRNAs in lipid nanoparticles have been administered in combination with antihistamines, non-steroidal anti-inflammatory drugs, and glucocorticoids7. A well-defined means of delivery is to directly conjugate a bioactive ligand to the RNA that will allow it to enter the cell of interest10; and, perhaps the most clinically advanced example of this technique, is the conjugation of N-acetylgalactosamine (GalNAc), which targets the asialoglycoprotein receptor on hepatocytes, to siRNA11. This receptor-mediated uptake allows for lower dosing, than that required for the therapeutic delivery of unconjugated oligonucleotides7, which is an essential feature for regulators approval and certification12. Delivery to other cell types, such as muscle cells, can be accomplished by targeting antibodies or antibody fragments against cell-surface proteins known to be involved in intracellular transport13.
These new scientific developments have shown us the different ways we can target unhealthy cells; but, aside from safety and delivery related issues that occur in the clinic, the greatest problem for approval is often not clinical efficiency, but Chemistry, Manufacturing, and Control (CMC) of quality processes. Some of these challenges include determining critical quality attributes and critical process parameters, low transduction efficiency, assessment of potency, assurance of product sterility, process validation, stability, and production at multiple manufacturing sites.
For that reason, in 2018, the FDA issued the CMC Information for Human Gene Therapy Investigational New Drug Applications (INDs) guidance document, intended to serve as part of a modern, comprehensive framework for how to advance the field of gene therapy14. In order to deliver a safe and effective product, human gene therapies present many manufacturing challenges. Some of these challenges include the variability and complexity inherent in the components used to generate the final product; such as the source of cells (i.e., autologous or allogeneic); the potential for adventitious agent contamination; the need for aseptic processing; and, in the case of ex vivo genetically modified cell therapies, the inability to “sterilize” the final product because it contains living cells15. Distribution of these products can also be a challenge due to stability issues, and the frequently short dating period of many ex vivo genetically modified cell products, which may need release of the final product for administration to a patient, before certain test results are available14. The guidance applies to human gene therapies and to combination products that contain a human gene therapy in combination with a drug or device; and, provides sponsors with recommendations on how to provide sufficient CMC information to assure safety, identity, quality, purity, and strength/potency of investigational gene therapy products16. By providing CMC information in an IND, the sponsor commits to perform the manufacturing and testing of the investigational product as described in the submission; and, to review it during all phases of development to ensure product safety and manufacturing control14. As clinical development proceeds, and additional product knowledge and manufacturing experience accumulate, sponsors should submit information amendments to modify the CMC information already submitted in the IND; and do so, prior to any implementation16.
The US Food and Drug Administration (FDA) approved more than 40 marketing applications for novel therapeutics and mechanisms of action in 2017, reflecting an unexpected quick progress in the field6.
Europe and the US have very different legal and regulatory regimes for approving gene therapies, with the main difference being that the FDA oversees clinical trials, whereas the European Medicines Agency (EMA) does not17. To run a clinical trial in any of the 28 members of the European Union (EU), approval needs to be gained from a competent authority and from the ethics committee in the specific member state; and, there is also the requisite to get approval for using a genetically modified organism (GMO)12. In the EU, this rapidly growing area of therapeutics is denominated Advanced Therapy Medicinal Products (ATMPs), which are legalized under a specific valid regulation (i.e., No. 1394/2007, EC)12. They are based on new and highly innovative technologies, that specifically cover cell and gene therapy products and tissue-engineered goods18. The Committee for Advanced Therapies (CAT) at EMA was established to ensure that the relevant expertise is available in regulatory decision making to support and evaluate these products19; and, in order to optimize their development and assessment, several guidelines have been developed by the EMA in collaboration with relevant regulatory experts within the EU12. A significant proportion of the products are being developed in academic settings or by small/medium-sized enterprises (SMEs)20. There are many factors that contribute to the challenges in developing these products in order to achieve successful clinical use, with the major one being the successful translation of the non-clinical (NC) (also often referred as pre-clinical) evidence to achieve clinical relevance19. The NC investigation for ATMPs is recommended to start with a risk-based approach (RBA) in planning, which is a unique feature in the ATMP regulation21. RBA aims to enable planning for relevant experiments for establishment of safety and efficiency, including first-in-human trial, market authorization and post-authorization follow-up19. Risks and risk factors are specific for the product and intended clinical use; and, they should be carefully determined by an integrated approach between CMC/NC/Clinical experts6. The certification procedure involves the scientific evaluation of quality data and, when available, non-clinical data that SMEs have generated at any stage of the ATMP development process. The goal of the certification is to identify any potential issues early on, so that these can be addressed before the submission of a marketing-authorization application21. If the data are in line with the requirements as set out in Directive 2001/83/EC, a certificate will be issued stating compliance of data with requirements6. An expedited regulatory pathway, known as the PRIME (PRIority Medicines) scheme, was introduced by the EMA in 2016 to facilitate approval of medicines that target unmet medical needs12, and to speed up patient access to these critical therapies. As in the US, the sponsor needs to ensure that the significant CMC aspects can be delivered, in the appropriate timeframe, such that safety and quality are not compromised6.
Synthetic mRNAs have the potential to be the new pillar for protein replacement therapy; since they are engineered to replace mutated mRNAs and to be immunologically unobtrusive, highly stable, while maximizing protein expression. There is no specific guidance from the FDA or EMA for mRNA therapeutic products; but the increasing number of clinical trials conducted under EMA and FDA oversight, shows that regulators have accepted the approaches proposed by various organizations to demonstrate that products are safe and acceptable for testing in humans22. Interestingly, depending on whether RNA-based therapeutics are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively22. If targeting against infectious diseases, mRNA falls into the broad vaccine category of genetic immunogens, then many of the guiding principles that have been defined for DNA vaccines and gene therapy vectors can likely be applied to mRNA, with some adaptations to reflect the unique features of mRNA23. Approaches to deliver mRNA into the cellular cytoplasm safely and efficiently have been developed, so that two mRNA-based approaches replacing Vascular Endothelial Growth Factor (VEGF), and Cystic Fibrosis Trans-membrane conductance Regulator (CFTR) have now made it into clinical trials23. All enzymes and reaction components required for the Good Manufacturing Practice (GMP) production of mRNA can be obtained from commercial suppliers as synthesized chemicals or bacterially expressed, animal component-free reagents, thus avoiding safety concerns that surround the adventitious agents that plague the manufacture of cell-culture-based therapeutics24. For some mRNA platforms, removal of double-stranded RNA, and other contaminants is a critical step for the potency of the final product, due to stimulation of interferon-dependent translation inhibition10. The use of reverse-phase Fast Protein Liquid Chromatography (FPLC) has successfully resolved this issue at the laboratory scale25, and processes of scalable aqueous purification approaches are now being considered23.
It is still early days for gene therapies and mRNA therapeutics; but so far, developers generally give regulatory agencies high marks as partners. There are now so many mechanisms for interacting with sponsors, that regulators have been very supportive of innovation and gene therapy in general. Both researchers and regulators in the field, say the new challenges come from the novelty of the science, and not so much from the regulatory aspects17.
ABOUT THE AUTHOR Catarina Carrão, biochemist by degree, worked as a biomedical researcher at Max F. Perutz Laboratories, Yale Cardiovascular Center at Yale University School of Medicine, and the Center Cardiovascular Research (CCR) at Charité Medical University. She now runs BioSciPons and Countlesssheep.com, a science communication agency and blog.
References:
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