Malini Mukherjee

Background: Unintended particle formation in vaccine samples has the potential to reduce product efficacy and impact immunogenicity. As a result, regulatory agencies routinely request tests to analyze particles in the micron size range for release. Particles may come in a variety of forms and can be inherent (e.g., from proteinaceous aggregates) or intrinsic (e.g., silicone oil from tubing or contamination from the manufacturing process) and can range in size from submicron to the visible size range. In recent years, there has been an increased interest in developing methods to detect and monitor aggregated particles as well as a desire to evaluate particle size and concentration of submicron particles in addition to the subvisible ranges routinely monitored by compendial methods. Methods: Typically utilized techniques include biophysical analytical methodologies that leverage light scattering or imaging. In this study, dynamic light scattering (DLS), micro-flow imaging (MFI), and flow virometry were evaluated as methods to detect and monitor submicron- to micron-sized particles. Results: These methods enabled assessments of stability and processability for drug substance (DS) and drug product (DP) samples of a Human Cytomegalovirus (HCMV) vaccine candidate. For this attenuated live virus vaccine candidate, MFI and flow virometry proved to be the most sensitive methods to monitor aggregate formation over the complete size range of potential aggregates. These established methods were applied to a coxsackievirus A21 vaccine candidate for process and formulation screening. Conclusions: MFI was most sensitive to changes in subvisible particle concentration of samples that were stressed via constant stirring, while flow virometry captured changes in submicron particles after stress from stirring and recirculation with a shear cell. This comprehensive screening approach combining multiple analytical methods to screen the full aggregate particle size range coupled with a broad range of forced aggregation conditions provides a framework for screening and development of vaccine formulation candidates and other modalities, such as biologics.

The authors would like to make the following corrections to this published paper

The quantitation of L1 protein is an important metric during the pharmaceutical manufacturing of human papilloma virus (HPV) vaccines, as they are critical components of virus like proteins (VLPs) that form the core of the vaccine matrix. The L1 protein is present in multiple states, including monomer, multimer, capsomere, fully formed VLPs, as well as aggregate species, and their expression levels provides an important read-out of upstream productivity and downstream purification efficiency through the measurement of step yields. However, quantitation of total L1 is challenging not only due to its presence in multiple states, but also due to the matrix complexity and purification stage of the samples which spans complex lysate to cleaner post purification material. Current analytical methods typically implemented for L1 quantitation includes simple western, which has its advantages, but requires sensitive and robust antibodies and are highly dependent on binding to accessible and linear regions of the L1. Additionally, conventional one-dimensional separation of L1 is ineffective where L1 and host cell protein or cellular components coelute, particularly in high-complexity lysate samples and intermediates prior to chromatographic capture steps. Herein, we present a sample preparation strategy and analytical method that is capable of total L1 quantitation regardless of its multimeric state and is compatible with sample matrices ranging from crude lysate to purified samples, without the use of complicated and serotype-specific reagents. We employ reduction and heat-denaturation during sample preparation to simplify the multimeric states of L1 to its monomer form and utilize two-dimension liquid chromatography (2D-LC) with first dimension (1D) size exclusion and second dimension (2D) reversed phase separation modes and demonstrate a suitable method for total L1 quantitation in multiple process intermediate steps.

The growing use of adjuvants in the fast-paced formulation of new vaccines has created an unprecedented need for meaningful analytical assays that deliver reliable quantitative data from complex adjuvant and adjuvant-antigen mixtures. Due to their complex chemical and physical properties, method development for the separation of vaccine adjuvants is considered a highly challenging and laborious task. Reversed-phase liquid chromatography (RPLC) is among the most important tests in the (bio)pharmaceutical industry for release and stability indicating measurements including adjuvant content, identity, and purity profile. However, the time constraints of developing “on-demand” robust quantitative methods prior to each change in formulation can easily lead to sample analysis becoming a bottleneck in vaccine development. Herein a simple and efficient generic analytical framework capable of chromatographically resolving the most commonly used non-aluminum based adjuvants across academic and industrial sectors is introduced. This was designed to seek a more proactive approach for fast-paced assay development endeavors that evolved from extensive stationary phase screening in conjunction with multifactorial in silico simulations of adjuvant retention time (RT) as a function of gradient time, temperature, organic modifier blending, and buffer concentration. The multifactorial retention models yield 3D resolution maps with excellent baseline separation of all adjuvants in a single run, which was found to be very accurate, with differences between experimental and simulated retention times of less than 1%. The analytical framework described here also includes the introduction of a more versatile approach to method development by introducing a dynamic RT database for adjuvants covering the entire library of adjuvants with broad mechanisms of action across numerous vaccine formulations with excellent linearity, accuracy, precision, and specificity. The power of this framework was also demonstrated with numerous analytical assays that can be generated rapidly from simulations guiding vaccine processes in the development of new adjuvant formulations. Analytical assay in this work covers content, purity profile by RPLC-UV-CAD, and component identification (RPLC-MS) across complex vaccine formulations, including the use of surfactants (e.g., polysorbates), as well as their separation from adjuvant targets.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose–response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG–lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Vaccinations to prevent infectious diseases are given to target the body’s innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte’s Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD’s Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.