Full-length G glycoprotein directly extracted from rabies virus with detergent and then stabilized by amphipols in liquid and freeze-dried forms.

Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.

Stability Preparedness: The Not-So-Cold Case for Innovations in Vaccine Stability Modelling and Product Release.

The rapid development of equitably accessible vaccines is paramount in addressing emerging global health challenges. The safety and efficacy of vaccines hinge significantly on their ability to remain stable from manufacturing throughout the supply chain and up to administration. Furthermore, the release of vaccines requires sufficient understanding of the stability profile to allow for expiration dating. In the event of a public health crisis, the time to generate the necessary stability data and the need for rapid product release are in direct opposition. Developing manufacturing platforms with thermostable product formulations for rapid response is therefore key to meeting CEPI's 100 Days Mission goal. This Review aims to highlight the need for stability preparedness through developing thermostable vaccine platforms and exploring innovative stability monitoring strategies that leverage advanced technologies, predictive modelling, and adaptive methodologies. By doing so, we seek to enhance the efficiency and effectiveness of stability assessments, supporting rapid development, regulatory approval, and widespread, equal distribution of vaccines-especially in an outbreak scenario. Finally, enhanced thermostability will allow for simplification across the supply chain, which will reduce the financial burden of vaccination programmes and enhance equitable access.

Stability modeling methodologies to enable earlier patient access.

Over recent years, confidence has been gained that predictive stability modeling approaches using statistical tools, prior knowledge and industry experience enable, in many instances, a robust and reliable shelf-life/expiry or retest period prediction for medicinal products. These science and risk-based approaches can compensate for not having a complete real-time stability data set to be included in regulatory applications at the time of initial submission and, thereby, accelerate the availability of new medicines. Examples of predictive stability modeling include accelerated stability assessment procedure (ASAP), advanced kinetic modeling (AKM), and novel modeling approaches that involve the use of Bayesian statistics and Artificial Intelligence (AI) applications such as Machine Learning (ML), with applicability to both synthetic and biological molecules. For biologics, product-specific and platform prior knowledge could be used to overcome model limitations known for non-quantitative stability indicating attributes. A successful ongoing verification approach by comparing the predicted data with real-time stability data would be an appropriate risk management approach which is intended to address regulatory concerns, and further build confidence in the robustness of these predictive modelling approaches with regulatory agencies. Global regulatory acceptance of stability modeling could allow patients to receive potential life-saving medications faster without compromising quality, safety or efficacy.

Case Studies on Changes and Proposed Process Development Approaches reflecting applicability of PDA Technical Report No. 89 Strategies for Vaccine Development and Lifecycle Management.

Vaccines are complex and a very diverse group of products with relatively long product lifecycles. The manufacturing programs for these vaccines need to be continually updated to comply with evolving regulatory expectations. Members of the Parenteral Drug Association (PDA) Vaccines Interest Group (VIG) authored and published PDA Technical Report No. 89 Strategies for Vaccine Development and Lifecycle Management (TR 89) which seeks to provide context to vaccine developers and manufacturers regarding key aspects of new or legacy vaccines such as control strategy from process development to vaccine lifecycle management, comparability and lifecycle management including technical, validation, quality and regulatory perspectives. To further explain and illustrate the concepts and topics discussed, seven relevant situations were selected as either case studies associated with changes implemented or proposed process development strategies, which are discussed in this article. The situations described are: working cell bank, modification or update of externally supplied product contact components for vaccine manufacturing, raw material change, new product at an existing site, vaccine development acceleration by leveraging existing platforms, selection and implementation of potency method, and modeling for stability forecast prediction. For each situation, the applicable key concepts from TR#89 are discussed as follows: Control Strategy, Prior Knowledge, Relying on PQS, Classification of Parameters, Validation Approach, Use of a Risk-based Approach, Comparability, Use of ICHQ12 and Additional Regulatory Considerations.

Formulation Development of a COVID-19 Recombinant Spike Protein-Based Vaccine.

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen-deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein.

Long-Term Stability Prediction for Developability Assessment of Biopharmaceutics Using Advanced Kinetic Modeling.

A crucial aspect of pharmaceutical development is the demonstration of long-term stability of the drug product. Biopharmaceuticals, such as proteins or peptides in liquid formulation, are typically administered via parental routes and should be stable over the shelf life, which generally includes a storing period (e.g., two years at 5 °C) and optionally an in-use period (e.g., 28 days at 30 °C). Herein, we present a case study where chemical degradation of SAR441255, a therapeutic peptide, in different formulations in combination with primary packaging materials was analyzed under accelerated conditions to derive long-term stability predictions for the recommended storing conditions (two years at 5 °C plus 28 days at 30 °C) using advanced kinetic modeling. These predictions served as a crucial decision parameter for the entry into clinical development. Comparison with analytical data measured under long-term conditions during the subsequent development phase demonstrated a high prediction accuracy. These predictions provided stability insights within weeks that would otherwise take years using measurements under long-term stability conditions only. To our knowledge, such in silico studies on stability predictions of a therapeutic peptide using accelerated chemical degradation data and advanced kinetic modeling with comparisons to subsequently measured real-life long-term stability data have not been described in literature before.

How to accelerate the supply of vaccines to all populations worldwide? Part I: Initial industry lessons learned and practical overarching proposals leveraging the COVID-19 situation.

The COVID-19 pandemic has shown itself to be an unprecedented challenge for vaccines which are widely recognized as the most important tool to exit this pandemic. We have witnessed vaccine scientists, developers, manufacturers, and stakeholders deliver several vaccines in just about a year. This is an unprecedented achievement in an environment that was not ready to manage such a global public health crisis. Indeed, the pandemic has highlighted some hurdles that need to be addressed in the system in order to streamline the regulatory processes and be in a situation where life-saving pharmaceutical solutions such as vaccines can be delivered quickly and equitably to people across the globe. More precisely, trade-offs had to be made between the need for regulatory flexibility in the requirements for manufacturing and controls to enable rapid availability of large volumes of vaccines vs the increased stringency and the lack of harmonization in the regulatory environment for vaccines globally. It is also characterized by a high heterogeneity in terms of review and approval processes, limiting equitable and timely access. We review and highlight the challenges relating to several topics, including process validation, comparability, stability, post-approval-changes, release testing, packaging, genetically modified organisms and variants. We see four areas for accelerating access to vaccines which provide solutions for the regulatory concerns, (1) science- and risk-based approaches, (2) global regulatory harmonization, (3) use of reliance, work-sharing, and recognition processes and (4) digitalization. These solutions are not new and have been previously highlighted. In recent months, we have seen some progress at the health authority level, but still much needs to be done. It is now time to reflect on the first lessons learnt from a devastating pandemic to ultimately ensure quick and wide access to medicines and vaccines for the citizens and patients.

How to accelerate the supply of vaccines to all populations worldwide? Part II: Initial industry lessons learned and detailed technical reflections leveraging the COVID-19 situation.

Vaccine discovery and vaccination against preventable diseases are one of most important achievements of the human race. While medical, scientific & technological advancements have kept in pace and found their way into treatment options for a vast majority of diseases, vaccines as a prevention tool in the public health realm are found languishing in the gap between such innovations and their easy availability/accessibility to vulnerable populations. This paradox has been best highlighted during the unprecedented crisis of the COVID-19 pandemic. As part of a two series publication on the vaccine industry's view on how to accelerate the availability of vaccines worldwide, this paper offers a deep dive into detailed proposals to enable this objective. These first-of-its-kind technical proposals gleaned from challenges and learnings from the COVID-19 pandemic are applicable to vaccines that are already on the market for routine pathogens as well as for production of new(er) vaccines for emerging pathogens with a public health threat potential. The technical proposals offer feasible and sustainable solutions in pivotal areas such as process validation, comparability, stability, post-approval changes, release testing, packaging, genetically modified organisms and variants, which are linked to manufacturing and quality control of vaccines. Ultimately these proposals aim to ease high regulatory complexity and heterogeneity surrounding the manufacturing & distribution of vaccines, by advocating the use of (1) Science and Risk based approaches, (2) global regulatory harmonization, (3) use of reliance, work-sharing, and recognition processes and (4) digitalization. Capitalizing & collaborating on such new-world advancements into the science of vaccines will eventually benefit the world by turning vaccines into vaccination, ensuring the health of everyone.

Predictive modeling for assessing the long-term thermal stability of a new fully-liquid quadrivalent meningococcal tetanus toxoid conjugated vaccine.

Establishing product stability is critical for pharmaceuticals. We used a modeling approach to predict the thermal stability of a fully-liquid quadrivalent meningococcal (serogroups A, C, W, Y) conjugate vaccine (MenACYW-TT; MenQuadfi®) at potential transportation and storage temperatures. Vaccine degradation was determined by measuring the rate of hydrolysis through an increase of free polysaccharide (de-conjugated or unconjugated polysaccharide) content during six months storage at 25 °C, 45 °C and 56 °C. A procedure combining advanced kinetics and statistics was used to screen and compare kinetic models describing observed free polysaccharide increase as a function of time and temperature for each serogroup. Statistical analyses were used to quantify prediction accuracy. A two-step kinetic model described the increase in free polysaccharide content for serogroup A; whereas, one-step kinetic models were found suitable to describe the other serogroups. The models were used to predict free polysaccharide increases for each serogroup during long-term storage under recommended conditions (2-8 °C), and during temperature excursions to 25 °C or 40 °C. In both cases, serogroup-specific simulations accurately predict the respective observed experimental data. Experimental data collected to 48 months at 5 °C were within 99% predictive bands. The models described here can be used with confidence to establish shelf-life for this fully-liquid quadrivalent meningococcal conjugate vaccine; as well as, monitor in real-time free polysaccharide increase for vaccines experiencing temperature excursions during shipment/storage.

Use of Stability Modeling to Support Accelerated Vaccine Development and Supply.

Stability assessment of pharmaceuticals in specific storage and shipment conditions is a key requirement to ensure that safe and efficacious products are administered to patients. This is particularly relevant for vaccines, with numerous vaccines strictly requiring cold storage to remain stable. When stability evaluation is exclusively based on real-time data, it may represent a bottleneck for rapid and effective vaccine access. Stability modeling for vaccines represents a key resource to predict stability based on accelerated stability studies; nevertheless, this approach is not fully exploited for these kinds of products. This is likely because of the complexity and diversity of vaccines, as well as the limited availability of dedicated guidelines or illustrative case studies. This article reports a cross-company perspective on stability modeling for vaccines. Several examples, based on the direct experience of the contributors, demonstrate that modeling approaches can be highly valuable to predict vaccines' shelf life and behavior during shipment or manipulation. It is demonstrated that modeling methodologies need to be tailored to the nature of the vaccine, the available prior knowledge, and the monitored attributes. Considering that the well-established strategies reported in ICH or WHO guidelines are not always broadly applicable to vaccines, this article represents an important source of information for vaccine researchers and manufacturers, setting the grounds for further discussion within the vaccine industry and with regulators.

A spray freeze dried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate.

The stability of live-attenuated viruses is very challenging due to thermal sensitivity; therefore, solid form is usually required (often freeze-dried products). Micropellet technology is a lyophilization technology that has the potential to provide greater flexibility in the presentation of a given vaccine particularly in multi-dose format or in combination of different vaccines. As a novel vaccine alternative process, this spray freeze-dried (SFD) micropellet technology was evaluated using as a model a yellow fever virus produced in Vero cells (vYF). Screening of excipients was performed in order to optimize physico-chemical properties of the micropellets. Sugar/polymer-based formulations induced high glass transition temperature (Tg), adequate breaking force and attrition resistance of the SFD micropellets. These mechanical parameters and their stability are of considerable importance for the storage, the transport but also the filling process of the SFD micropellets. By adding excipients required to best preserve virus infectivity, an optimal sugar/polymer-based formulation was selected to build micropellets containing vYF. Monodisperse and dried micropellets with a diameter of about 530 µm were obtained, exhibiting similar potency to conventional freeze-dried product in terms of vYF infectious titer when both solid forms were kept under refrigerated conditions (2-8 °C). Comparable kinetics of degradation were observed for vYF formulated in micropellets or as conventional freeze-dried product during an accelerated stability study using incubations at 25 °C and 37 °C over several weeks. The results from this investigation demonstrate the ability to formulate live-attenuated viruses in micropellets. Pharmaceutical applications of this novel vaccine solid form are discussed.

Accurate prediction of vaccine stability under real storage conditions and during temperature excursions.

Due to their thermosensitivity, most vaccines must be kept refrigerated from production to use. To successfully carry out global immunization programs, ensuring the stability of vaccines is crucial. In this context, two important issues are critical, namely: (i) predicting vaccine stability and (ii) preventing product damage due to excessive temperature excursions outside of the recommended storage conditions (cold chain break). We applied a combination of advanced kinetics and statistical analyses on vaccine forced degradation data to accurately describe the loss of antigenicity for a multivalent freeze-dried inactivated virus vaccine containing three variants. The screening of large amounts of kinetic models combined with a statistical model selection approach resulted in the identification of two-step kinetic models. Predictions based on kinetic analysis and experimental stability data were in agreement, with approximately five percentage points difference from real values for long-term stability storage conditions, after excursions of temperature and during experimental shipments of freeze-dried products. Results showed that modeling a few months of forced degradation can be used to predict various time and temperature profiles endured by vaccines, i.e. long-term stability, short time excursions outside the labeled storage conditions or shipments at ambient temperature, with high accuracy. Pharmaceutical applications of the presented kinetics-based approach are discussed.

Biophysical virus particle specific characterization to sharpen the definition of virus stability.

Vaccine thermostability is key to successful global immunization programs as it may have a significant impact on the continuous cold-chain maintenance logistics, as well as affect vaccine potency. Modern biological and biophysical techniques were combined to in-depth characterize the thermostability of a formulated rabies virus (RABV) in terms of antigenic and genomic titer, virus particle count and aggregation state. Tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA) were used to count virus particles while simultaneously determining their size distribution. RABV antigenicity was assessed by NTA using a monoclonal antibody that recognize a rabies glycoprotein (G protein) conformational epitope, enabling to specifically count antigenic rabies viruses. Agreement between antigenicity results from NTA and conventional method, as ELISA, was demonstrated. Additionally, NTA and ELISA showed mirrored loss of RABV antigenicity during forced degradation studies performed between 5 °C and 45 °C temperature exposure for one month. Concomitant with decreased antigenicity, emergence of RABV particle populations larger than those expected for rabies family viruses was observed, suggesting RABV aggregation induced by thermal stress. Finally, using a kinetic-based modeling approach to explore forced degradation antigenicity data (NTA, ELISA), a two-step model accurately describing antigenicity loss was identified. This model predicted a RABV shelf-life of more than 3 years at 5 °C; significant loss of antigenicity was predicted for samples maintained several months at ambient temperature. This thorough characterization of RABV forced degradation study originally provided a time-temperature mapping of RABV stability.

Advanced kinetic analysis as a tool for formulation development and prediction of vaccine stability.

We have used a protein-based vaccine, a live virus vaccine, and an experimental adjuvant to evaluate the utility of an advanced kinetic modeling approach for stability prediction. The modeling approach uses a systematic and simple procedure for the selection of the most appropriate kinetic equation to describe the degradation rate of compounds subjected to accelerated conditions. One-step and two-step reactions with unlimited combinations of kinetic models were screened for the three products under evaluation. The most appropriate mathematical model for a given product was chosen based on the values of residual sum of squares and the weight parameter w. A relatively simple n-th order kinetic model best fitted the degradation of an adjuvanted protein vaccine with a prediction error lower than 10%. A more complex two-step model was required to describe inactivation of a live virus vaccine under normal and elevated storage temperatures. Finally, an autocatalytic-type kinetic model best fitted the degradation of an oil-in-water adjuvant formulation. The modeling approach described here could be used for vaccine stability prediction, expiry date estimation, and formulation selection. To the best of our knowledge, this is the first report describing a global kinetic analysis of degradation of vaccine components with high prediction accuracy.