Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran) and mRNA (COVID-19 vaccine) drugs

Nucleic acid therapeutics are developing into precise medicines that can manipulate specific genes. However, the development of safe and effective delivery system for the target cells has remained a challenge. Lipid nanoparticles (LNPs) have provided a revolutionary delivery system that can ensure multiple clinical translation of RNA-based candidates. In 2018, Patisiran (Onpattro) was first approved as an LNP-based siRNA drug. In 2020, during the coronavirus disease 2019 (COVID-19) outbreak, LNPs have enabled the development of two SARS-CoV-2 mRNA vaccines, Tozinameran (Comirnaty or Pfizer-BioNTech COVID-19 vaccine) and Elasomeran (Spikevax or COVID-19 vaccine Moderna) for conditional approval. Here, we reviewed the state-of-the-art LNP technology employed in three approved drugs (one siRNA-based and two mRNA-based drugs) and discussed the differences in their mode of action, formulation design, and biodistribution.     

Addressing the Cold Reality of mRNA Vaccine Stability

As mRNA vaccines became the frontrunners in late-stage clinical trials to fight the COVID-19 pandemic, challenges surrounding their formulation and stability became readily apparent. In this commentary, we first describe company proposals, based on available public information, for the (frozen) storage of mRNA vaccine drug products across the vaccine supply chain. We then review the literature on the pharmaceutical stability of mRNA vaccine candidates, including attempts to improve their stability, analytical techniques to monitor their stability, and regulatory guidelines covering product characterization and storage stability. We conclude that systematic approaches to identify the key physicochemical degradation mechanism(s) of formulated mRNA vaccine candidates are currently lacking. Rational design of optimally stabilized mRNA vaccine formulations during storage, transport, and administration at refrigerated or ambient temperatures should thus have top priority in the pharmaceutical development community. In addition to evidence of human immunogenicity against multiple viral pathogens, including compelling efficacy results against COVID-19, another key strength of the mRNA vaccine approach is that it is readily adaptable to rapidly address future outbreaks of new emerging infectious diseases. Consequently, we should not wait for the next pandemic to address and solve the challenges associated with the stability and storage of formulated mRNA vaccines.     

Stress Factors in Primary Packaging,Transportation and Handling of Protein Drug Products and Their Impact on Product Quality

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality.     

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.     

强制降解试验在药物研发中的应用

目的 阐述强制降解试验研究现状,汇总各强制降解试验基本条件,为药物研发提供借鉴和参考。方法 汇总实际工作和文献中药物降解基本方法,分析药品注册申报各阶段对降解研究的要求。结果 现阶段各国监管机构没有提出统一明确的强制降解试验条件,药物的降解条件与药物分子结构及制剂处方相关。结论 强制降解研究可以帮助确定药物的降解途径并解析降解产物,为药物安全性和毒理学研究提供支持,也为稳定性指示分析方法、药物处方、包装选择和储存条件的开发提供支持。从药物研发早期到后期,强制降解需要不断完善。     

The role of degradant profiling in active pharmaceutical ingredients and drug products

Forced degradation studies are used to facilitate the development of analytical methodology, to gain a better understanding of active pharmaceutical ingredient (API) and drug product (DP) stability, and to provide information about degradation pathways and degradation products. In order to fulfill development and regulatory needs, this publication provides a roadmap for when and how to perform studies, helpful tools in designing rugged scientific studies, and guidance on how to record and communicate results.     

Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles.

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed.     

Protein Stability After Administration: A Physiologic Consideration

Regulatory authorities and the scientific community have identified the need to monitor the in vivo stability of therapeutic proteins (TPs). Due to the unique physiologic conditions in patients, the stability of TPs after administration can deviate largely from their stability under drug product (DP) conditions. TPs can degrade at substantial rates once immersed in the in vivo milieu. Changes in protein stability upon administration to patients are critical as they can have implications on patient safety and clinical effectiveness of DPs.Physiologic conditions are challenging to simulate and require dedicated in vitro models for specific routes of administration. Advancements of in vitro models enable to simulate the exposure to physiologic conditions prior to resource demanding pre-clinical and clinical studies. This enables to evaluate the in vivo stability and thus may allow to improve the safety/efficacy profile of DPs. While in vitro-in vivo correlations are challenging, benchmarking DP candidates enables to identify liabilities and optimize molecules. The in vivo stability should be an integral part of holistic stability assessments during early development. Such assessments can accelerate development timelines and lead to more stable DPs for patients.     

A Rapid High-Sensitivity Reversed–Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.     

Stability of peptides and therapeutic success in cancer

INTRODUCTION: Although naturally occurring peptides have been widely used as drugs, their rapid in vivo degradation by proteolysis and their interactions at multiple receptors are part of the reason for the limitation of their clinical applications. AREAS COVERED: This paper reviews peptide-metabolizing enzymes in the brain and intestinal brush-border membranes, and discusses potential strategies to improve biological activity, specificity and stability of peptides. The reader will gain, via some examples, an appreciation of the challenges involved in identifying peptides stability to improve their biological properties such as selectivity. EXPERT OPINION: Due to the metabolic process, it is crucial to follow the biodistribution of a peptide drug and/or a peptidic moiety in order to improve its biological properties such as selectivity. To these purposes, pseudopeptides and peptidomimetics preserving the biological properties of native peptides have been developed to increase their resistance to degradation and elimination, bioavailability and selectivity to become good drug candidates.     

pH control of nucleophilic/electrophilic oxidation

Finding formulations that prevent degradation of the active pharmaceutical ingredient is an essential part of drug development. One of the major mechanisms of degradation is oxidation. Oxidative degradation is complex, and can occur via different mechanisms, such as autoxidation, nucleophilic/electrophilic addition, and electron transfer reactions. This paper uses three model compounds and determines the mechanisms of oxidation and strategies to reduce degradation. The mechanism of oxidation was established by comparing the results of different forced degradation experiments (radical initiation and peroxide addition), computational chemistry to those of formulated drug product stability. The model compounds chosen contained both oxidizable amine and sulfide functional groups. Although, both oxidative forced degradation conditions showed different impurity profiles the peroxide results mirrored those of the actual stability results of the drug product. The major degradation pathway of all compounds tested was nucleophilic/electrophilic oxidation of the amine to form N-oxide. Strategies to prevent this oxidation were explored by performing forced degradation experiments of the active pharmaceutical ingredient (API) in solution, in slurries containing standard excipient mixtures, and in solid formulation blends prepared by wet granulation. The reaction was significantly influenced by pH in solvent and excipient slurries, with 100% degradation occurring at basic pH values (>pH 8) and no degradation occurring at pH 2 upon exposure to 0.3% peroxide. Wet granulated blends were also stabilized by lowering the pH during granulation through the addition of citric acid prior to the solution of peroxide, resulting in little (0.02% maximum) or no degradation for the four different blends after 6 week storage at 40 °C/75%RH.     

Analytical and Preformulation Characterization Studies of Human Papillomavirus Virus-Like Particles to Enable Quadrivalent Multi-Dose Vaccine Formulation Development

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed. The four monovalent AH-adsorbed HPV VLPs were then characterized using these down-selected methods. Second, stability-indicating competitive ELISA assays were developed using HPV serotype-specific neutralizing mAbs, to monitor relative antibody binding profiles of the four AH-adsorbed VLPs during storage. Third, concentration-dependent preservative-induced destabilization of HPV16 VLPs was demonstrated by addition of eight preservatives found in parenterally administered pharmaceuticals and vaccines, as measured by ELISA, dynamic light scattering, and differential scanning calorimetry. Finally, preservative stability and effectiveness in the presence of vaccine components were evaluated using a combination of RP-UHPLC, a microbial growth inhibition assay, and a modified version of the European Pharmacopoeia assay (Ph. Eur. 5.1.3). Results are discussed in terms of analytical challenges encountered to identify and develop high-throughput methods that facilitate multi-dose formulation development of aluminum-adjuvanted protein-based vaccine candidates.     

Degradation pathways,analytical characterization and formulation strategies of a peptide and a protein calcitonine and human growth hormone in comparison

Peptides and proteins differ from conventional chemical entities in their sensitivity to numerous environmental factors and their susceptibility to different degradation pathways. Therefore, complex analytical methodologies are necessary to characterize their molecular entity as well as to detect and quantify the possible degradation products. The formulation of these molecules for a pharmaceutical product requires stabilization by various excipients. Most of the products are brought to market as solutions or lyophilisates. In the first part, this article presents a comparison between the degradation profile of a peptide (calcitonine) and a protein (human growth hormone), in solution and as a freeze-dried product. The various analytical methods used to characterize and identify the degradation products are reviewed and discussed. The second part contains an overview of the different formulation strategies for calcitonine and human growth hormone. Finally, the different stress conditions used to obtain stability data are discussed critically. This leads on to general comments on the design of stability studies for peptide and protein drugs as pharmaceuticals taking into consideration the official guidelines.     

Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies

The formulation development of monoclonal antibodies is extremely challenging, due to the diversity and complexity contained within this class of molecules. The physical and chemical properties of a monoclonal antibody dictate the behavior of the protein drug during manufacturing, storage and clinical administration. In the past few years, the use of high throughput technologies has been widely adapted to delineate unique properties of individual immunoglobulin G's (IgG's) important for their development. Numerous screening techniques have been designed to reveal physical and chemical characteristics of a protein relevant to stability under production, formulation and delivery conditions. In addition, protein stability under accelerated stresses has been utilized to predict long-term storage behavior for monoclonal antibodies in the formulation. In this review, we summarize the recent advancements in the field of biophysical technology, with a specific focus on the techniques that can be directly applied to the formulation development of monoclonal antibodies. Several case studies are also presented here to provide examples of combining existing biophysical methods with high throughput screening technology in the formulation development of monoclonal antibody drugs.     

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol® NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structues of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy- and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and Δ1, 10-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnosite tool for new degradation products.     

Influence of formulation technique for hydroxypropyl-beta-cyclodextrin on the stability of aspirin in HFA 134a.

The objective of this study was to determine the influence of the formulation technique for 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) on the stability of aspirin in a suspension-based pressurized metered-dose inhaler (pMDI) formulation containing a hydrofluoroalkane (HFA) propellant. HP beta CD was formulated in a pMDI as a lyophilized inclusion complex or a physical mixture with aspirin. A pMDI formulation containing aspirin alone was used as the control. The chemical stability of aspirin in each pMDI formulation was determined over 6-months storage at 5, 25 and 40 degrees C. The quantity of water taken up into the pMDI canister was determined by Karl Fisher titration after storage for 6 months. Differential scanning calorimetry (DSC) was used to confirm the formation of a complex between HP beta CD and aspirin. Aspirin in the lyophilized inclusion complex exhibited the most significant degree of degradation during the 6-months storage, while aspirin alone in the pMDI demonstrated a moderate degree of degradation. Aspirin formulated in the physical mixture displayed the least degree of degradation. The water uptake study showed that water ingress occurred to the greatest extent for formulations containing aspirin and HP beta CD physical mixture, and to the least extent for formulations containing aspirin alone. Finally, the DSC study indicated that an inclusion complex was formed in situ in the pMDI formulations containing the HP beta CD and aspirin physical mixture. In conclusion, HP beta CD may be used to enhance the stability of a chemically labile drug, but the drug stability may be affected by the method of preparation of the formulation.     

A Systematic Multitechnique Approach for Detection and Characterization of Reversible Self-Association during Formulation Development of Therapeutic Antibodies

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self‐association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development. Considering the large number of available analytical methods, choice of the employed technique is an important contributing factor for successful investigation of RSA. Herein, a multitechnique (dynamic light scattering, multiangle static light scattering, and analytical ultracentrifugation) approach is employed to comprehensively characterize the self‐association of a model immunoglobulin G1 molecule. Studies herein discuss an effective approach for detection and characterization of RSA during biopharmaceutical development based on the capabilities of each technique, their complementarity, and more importantly their suitability for the stage of development in which RSA is investigated.     

Ongoing Challenges to Develop High Concentration Monoclonal Antibody-based Formulations for Subcutaneous Administration: Quo Vadis?

Although many subcutaneously (s.c.) delivered, high-concentration antibody formulations (HCAF) have received regulatory approval and are widely used commercially, formulation scientists are still presented with many ongoing challenges during HCAF development with new mAb and mAb-based candidates. Depending on the specific physicochemical and biological properties of a particular mAb-based molecule, such challenges vary from pharmaceutical attributes e.g., stability, viscosity, manufacturability, to clinical performance e.g., bioavailability, immunogenicity, and finally to patient experience e.g., preference for s.c. vs. intravenous delivery and/or preferred interactions with health-care professionals. This commentary focuses on one key formulation obstacle encountered during HCAF development: how to maximize the dose of the drug? We examine methodologies for increasing the protein concentration, increasing the volume delivered, or combining both approaches together. We discuss commonly encountered hurdles, i.e., physical protein instability and solution volume limitations, and we provide recommendations to formulation scientists to facilitate their development of s.c. administered HCAF with new mAb-based product candidates.     

Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that “stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution”. Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs). Hastelloy®-based metal containers are sometimes used due to their higher corrosion resistance and significantly lower iron content to mitigate the potential corrosion-related risks associated with high salt formulations. Despite their benefits, we have found that elevated temperature stability studies in small scale Hastelloy® containers can lead to degradation that is not representative of degradation under typical storage conditions relevant to the manufacturing process. We provide evidence for an oxidation-induced aggregation mechanism that is based on Fenton chemistry with peroxide being supplied by the autoxidation of polysorbate at stress temperatures. Further, variation in the rates of iron leaching between individual small scale containers is shown to be the cause of the variable rates of degradation through strong correlations between leached iron levels and the extents of oxidation and aggregation. The addition of a metal chelator or the removal of polysorbate from the formulation mitigates the oxidation and the non-representative behavior. Extended characterization by LC-MS and ¹⁸O labeled peptide mapping shows that a significant portion of the aggregate formed under these conditions is covalently crosslinked and that the predominant covalent species is either a dityrosine or tyrosine-tryptophan crosslink between an Fc peptide and a Fab peptide. This report is the first time either of these two crosslinks have been reported for antibodies with detailed analytical characterization. Because the behavior observed in these studies is not representative of degradation under typical storage conditions relevant to the manufacturing process, this study demonstrates that small scale stress studies in metal containers should be performed with caution and that extended incubation times can lead to non-representative degradation mechanisms.