ABSTRACT
The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.
ABSTRACT
Pharmaceutical product development guided by Quality by Design (QbD) is based on a complete understanding of the critical process parameters (CPPs) that are important for achieving the desired product critical quality attributes (CQAs). The effect of process settings, such as the screw speed, the throughput, the barrel temperature, and the screw configuration, is a well-known factor in the setup of pharmaceutical hot melt extrusion (HME) processes. A CPP that has not yet been extensively researched is the type of cross-section geometry of the screw elements. Typically, pharmaceutical extruders have double-flighted screw cross-sections, with some elements having a single- or triple-flighted element section. The exception is a NANO16 extruder from Leistritz, with all screw elements having a triple-flighted screw geometry. We investigated the process setup and scale-up to a double-flighted extruder experimentally and in silico via a digital twin. Two formulations were processed on a NANO16 extruder and virtually transferred to a ZSE18 double-flighted co-rotating twin-screw extruder. Detailed smoothed particle hydrodynamics simulations of all screw elements available from both extruders were performed, and their efficiency in conveying, pressure build-up, and power consumption were studied. Reduced-order 1D HME simulations, which were carried out to investigate the process space and scalability of both extruders, were experimentally validated.
Subject(s)
Hot Melt Extrusion Technology , Hot Temperature , Drug Compounding , Temperature , Pharmaceutical Preparations , Technology, PharmaceuticalABSTRACT
A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.
ABSTRACT
Small-sized temperature indicator additives autonomously record temperature events via a gradual irreversible signal change. This permits, for instance, the indication of possible cold-chain breaches or failure of electronics but also curing of glues. Thus, information about the materials' thermal history can be obtained upon signal detection at every point of interest. In this work, maximum-temperature indicators with magnetic readout based on micrometer-sized supraparticles (SPs) are introduced. The magnetic signal transduction is, by nature, independent of the materials' optical properties. This facilitates the determination of valuable temperature information from the inside, that is, the bulk, even of dark and opaque macroscopic objects, which might differ from their surface. Compared to state-of-the-art optical temperature indicators, complementary magnetic readout characteristics ultimately expand their applicability. The conceptualized SPs are hierarchically structured assemblies of environmentally friendly, inexpensive iron oxide nanoparticles and thermoplastic polymer. Irreversible structural changes, induced by polymer softening, yield magnetic interaction changes within and between the hierarchic sub-structures, which are distinguishable and define the temperature indication mechanism. The fundamental understanding of the SPs' working principle enables customization of the particles' working range, response time, and sensitivity, using a toolbox-like manufacturing approach. The magnetic signal change is detected self-referenced, fast, and contactless.
ABSTRACT
This paper presents a rational workflow for developing enabling formulations, such as amorphous solid dispersions, via hot-melt extrusion in less than a year. First, our approach to an integrated product and process development framework is described, including state-of-the-art theoretical concepts, modeling, and experimental characterization described in the literature and developed by us. Next, lab-scale extruder setups are designed (processing conditions and screw design) based on a rational, model-based framework that takes into account the thermal load required, the mixing capabilities, and the thermo-mechanical degradation. The predicted optimal process setup can be validated quickly in the pilot plant. Lastly, a transfer of the process to any GMP-certified manufacturing site can be performed in silico for any extruder based on our validated computational framework. In summary, the proposed workflow massively reduces the risk in product and process development and shortens the drug-to-market time for enabling formulations.
