Search Results (21)

Background/Objectives: Filamentous fungi, in particular the species Aspergillus, Scedosporium, and Exophiala, frequently colonize the lungs of cystic fibrosis (CF) patients. Chronic colonization is linked to hypersensitivity reactions and persistent infections leading to a significant long-term decline in lung function. Azole antifungal therapy such as voriconazole (VRC) slows disease progression, particularly in patients with advanced CF; however, excessive mucus production in CF lungs poses a diffusional barrier to effective treatment. Methods: Here, biodegradable nanohydrogels (NHGs) recently developed as nanocarriers were evaluated for formulating VRC as a platform for treating fungal infections in CF lungs. The NHGs entrapped up to about 30 μg/mg of VRC, and physicochemical properties were investigated via dynamic laser light scattering and nanoparticle tracking analysis. Diameters were 100–400 nm, and excellent colloidal stability was demonstrated in interstitial fluids, indicating potential for pulmonary delivery. Nano-formulations exhibited high in vitro cytocompatibility in A549 and HEK293T cells and were tested for the release of VRC under two different sink conditions. Results: Notably, the antifungal activity of VRC-loaded nanohydrogels was up to eight-fold greater than an aqueous suspension drug against different fungal species isolated from CF sputum, regardless of the presence of a CF artificial mucus layer. Conclusions: These findings support the development of potent VRC nano-formulations for treating fungal disorders in CF lungs. Full article

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 10⁹ TCID50/mL) in the culture supernatant, the final formulations were prepared at 10¹⁰ TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented. Full article

Major efforts in the intensification of cell culture-based viral vaccine manufacturing focus on the development of high-cell-density (HCD) processes, often operated in perfusion. While perfusion operations allow for higher viable cell densities and volumetric productivities, the high perfusion rates (PR) normally adopted—typically between 2 and 4 vessel volumes per day (VVD)—dramatically increase media consumption, resulting in a higher burden on the cell retention device and raising challenges for the handling and disposal of high volumes of media. In this study, we explore high inoculum fed-batch (HIFB) and low-PR perfusion operations to intensify a cell culture-based process for influenza virus production while minimizing media consumption. To reduce product retention time in the bioreactor, produced viral particles were continuously harvested using a tangential flow depth filtration (TFDF) system as a cell retention device and harvest unit. The feeding strategies developed—a hybrid fed-batch with continuous harvest and a low-PR perfusion—allowed for infections in the range of 8–10 × 10⁶ cells/mL while maintaining cell-specific productivity comparable to the batch control, resulting in a global increase in the process productivity. Overall, our work demonstrates that feeding strategies that minimize media consumption are suitable for large-scale influenza vaccine production. Full article

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein. Full article

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA. Full article

Research on platinum-based anticancer drugs continuously strives to develop new non-classical platinum complexes. Pt(IV) prodrugs are the most promising, and their activation-by-reduction mechanism of action is being explored as a prospect for higher selectivity and efficiency. Herein, we present the anticancer potency and chemical reactivity of Pt(IV) complexes formed by linking pyrene butyric acid with cisplatin. The results from cytotoxicity screening on 10 types of cancer cell lines and non-malignant cells (HEK-293) indicated IC₅₀ values as low as 50–70 nM for the monosubstituted Pt(IV) complex against leukemia cell lines (HL-60 and SKW3) and a cisplatin-resistant derivative (HL-60/CDDP). Interestingly, the bis-substituted complex is virtually non-toxic to both healthy and cancerous cells of adherent types. Nevertheless, it shows high cytotoxicity against multidrug-resistant derivatives HL-60/CDDP and HL-60/Dox. The reactivity of the complexes with biological reductants was monitored by the NMR method. Furthermore, the platinum uptake by the treated cells was examined on two types of cellular cultures: adherent and suspension growing, and proteome profiling was conducted to track expression changes of key apoptosis-related proteins in HL-60 cells. The general conclusion points to a possible cytoskeletal entrapment of the bulkier bis-pyrene complex that could be limiting its cytotoxicity to adherent cells, both cancerous and healthy ones. Full article

Melanin is an insoluble, amorphous polymer that forms planar sheets that aggregate naturally to create colloidal particles with several biological functions. Based on this, here, a preformed recombinant melanin (PRM) was utilized as the polymeric raw material to generate recombinant melanin nanoparticles (RMNPs). These nanoparticles were prepared using bottom-up (nanocrystallization—NC, and double emulsion–solvent evaporation—DE) and top-down (high-pressure homogenization—HP) manufacturing approaches. The particle size, Z-potential, identity, stability, morphology, and solid-state properties were evaluated. RMNP biocompatibility was determined in human embryogenic kidney (HEK293) and human epidermal keratinocyte (HEKn) cell lines. RMNPs prepared by NC reached a particle size of 245.9 ± 31.5 nm and a Z-potential of −20.2 ± 1.56 mV; 253.1 ± 30.6 nm and −39.2 ± 0.56 mV compared to that obtained by DE, as well as RMNPs of 302.2 ± 69.9 nm and −38.6 ± 2.25 mV using HP. Spherical and solid nanostructures in the bottom-up approaches were observed; however, they were an irregular shape with a wide size distribution when the HP method was applied. Infrared (IR) spectra showed no changes in the chemical structure of the melanin after the manufacturing process but did exhibit an amorphous crystal rearrangement according to calorimetric and PXRD analysis. All RMNPs presented long stability in an aqueous suspension and resistance to being sterilized by wet steam and ultraviolet (UV) radiation. Finally, cytotoxicity assays showed that RMNPs are safe up to 100 μg/mL. These findings open new possibilities for obtaining melanin nanoparticles with potential applications in drug delivery, tissue engineering, diagnosis, and sun protection, among others. Full article

HEK293 is a widely used cell line in the fields of research and industry. It is assumed that these cells are sensitive to hydrodynamic stress. The aim of this research was to use particle image velocimetry validated computational fluid dynamics (CFD) to determine the hydrodynamic stress in both shake flasks, with and without baffles, and in stirred Minifors 2 bioreactors to evaluate its effect on the growth and aggregate size distribution of HEK293 suspension cells. The HEK FreeStyle^TM 293-F cell line was cultivated in batch mode at different specific power inputs (from 63 $\mathrm{W} {\mathrm{m}}^{-3}$ to 451 $\mathrm{W} {\mathrm{m}}^{-3}$), whereby $\approx 60 \mathrm{W} {\mathrm{m}}^{-3}$ corresponds to the upper limit, which is what has been typically described in published experiments. In addition to the specific growth rate and maximum viable cell density VCD${}_{\max }$, the cell size distribution over time and cluster size distribution were investigated. The VCD${}_{\max }$ of $(5.77\pm 0.02)·{10}^6$$\mathrm{cells}$$\mathrm{m}$${\mathrm{L}}^{-1}$ was reached at a specific power input of 233 $\mathrm{W} {\mathrm{m}}^{-3}$ and was $23.8\%$ higher than the value obtained at 63 $\mathrm{W} {\mathrm{m}}^{-3}$ and $7.2\%$ higher than the value obtained at 451 $\mathrm{W} {\mathrm{m}}^{-3}$. No significant change in the cell size distribution could be measured in the investigated range. It was shown that the cell cluster size distribution follows a strict geometric distribution whose free parameter p is linearly dependent on the mean Kolmogorov length scale. Based on the performed experiments, it has been shown that by using CFD-characterised bioreactors, the VCD${}_{\max }$ can be increased and the cell aggregate rate can be precisely controlled. Full article

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries. Full article

Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI. Full article

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires’ disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-Blue^TM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion. Full article

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 10⁹ VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100–200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production. Full article

Adeno-associated virus vectors (AAV) are reported to have a great potential for gene therapy, however, a major bottleneck for this kind of therapy is the limitation of production capacity. Higher specific AAV vector yield is often reported for adherent cell systems compared to cells in suspension, and a microcarrier-based culture is well established for the culture of anchored cells on a larger scale. The purpose of the present study was to explore how microcarrier cultures could provide a solution for the production of AAV vectors based on the triple plasmid transfection of HEK293T cells in a stirred tank bioreactor. In the present study, cells were grown and expanded in suspension, offering the ease of this type of operation, and were then anchored on microcarriers in order to proceed with transfection of the plasmids for transient AAV vector production. This process was developed in view of a bioreactor application in a 200 mL stirred-tank vessel where shear stress aspects were studied. Furthermore, amenability to a continuous process was studied. The present investigation provided a proof-of-concept of a continuous process based on microcarriers in a stirred-tank bioreactor. Full article

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293^TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial. Full article

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID₅₀ and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 10⁸ TCID₅₀/mL for NDV-GFP and 1.33 × 10⁸ TCID₅₀/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 10⁸ TCID₅₀/mL for NDV-GFP and 3.16 × 10⁷ TCID₅₀/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets. Full article