Development and qualification of 3 L scale-down model for large scale vaccine process on vero cell culture using microcarriers.

Scale-down models (SDM) are pivotal tools for process understanding and improvement to accelerate the development of vaccines from laboratory research to global commercialization. In this study, a 3 L SDM representing a 50 L scale Vero cell culture process of a live-attenuated virus vaccine using microcarriers was developed and qualified based on the constant impeller power per volume principle. Both multivariate data analysis (MVDA) and the traditional univariate data analysis showed comparable and equivalent cell growth, metabolic activity, and product quality results across scales. Computational fluid dynamics simulation further confirmed similar hydrodynamic stress between the two scales.

Development and scale-up of rVSV-SARS-CoV-2 vaccine process using single use bioreactor.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the Coronavirus Disease 2019 (COVID-19) has spread through the globe at an alarming speed. The disease has become a global pandemic affecting millions of people and created public health crises worldwide. Among many efforts to urgently develop a vaccine against this disease, we developed an industrial-scale closed, single use manufacturing process for V590, a vaccine candidate for SARS-CoV-2. V590 is a recombinant vesicular stomatitis virus (rVSV) genetically engineered to express SARS-CoV-2 glycoprotein. In this work, we describe the development and optimization of serum-free microcarrier production of V590 in Vero cells in a closed system. To achieve the maximum virus productivity, we optimized pH and temperature during virus production in 3 liters (L) bioreactors. Virus productivity was improved (by ∼1 log) by using pH 7.0 and temperature at 34.0 °C. The optimal production condition was successfully scaled up to a 2000 L Single Use Bioreactor (SUB), producing a maximum virus titer of ∼1.0e+7 plaque forming units (PFU)/mL. Further process intensification and simplification, including growing Vero cells at 2 gs per liter (g/L) of Cytodex-1 Gamma microcarriers and eliminating the media exchange (MX) step prior to infection helped to increase virus productivity by ∼2-fold.

Design of a novel automated methanol feed system for pilot-scale fermentation of Pichia pastoris.

Large-scale fermentation of Pichia pastoris requires a large volume of methanol feed during the induction phase. However, a large volume of methanol feed is difficult to use in the processing suite because of the inconvenience of constant monitoring, manual manipulation steps, and fire and explosion hazards. To optimize and improve safety of the methanol feed process, a novel automated methanol feed system has been designed and implemented for industrial fermentation of P. pastoris. Details of the design of the methanol feed system are described. The main goals of the design were to automate the methanol feed process and to minimize the hazardous risks associated with storing and handling large quantities of methanol in the processing area. The methanol feed system is composed of two main components: a bulk feed (BF) system and up to three portable process feed (PF) systems. The BF system automatically delivers methanol from a central location to the portable PF system. The PF system provides precise flow control of linear, step, or exponential feed of methanol to the fermenter. Pilot-scale fermentations with linear and exponential methanol feeds were conducted using two Mut(+) (methanol utilization plus) strains, one expressing a recombinant therapeutic protein and the other a monoclonal antibody. Results show that the methanol feed system is accurate, safe, and efficient. The feed rates for both linear and exponential feed methods were within ± 5% of the set points, and the total amount of methanol fed was within 1% of the targeted volume.

Neurotoxicity assessment using zebrafish.

INTRODUCTION: Transparency is a unique attribute of zebrafish that permits direct assessment of drug effects on the nervous system using whole mount antibody immunostaining and histochemistry. METHODS: To assess pharmacological effects of drugs on the optic nerves, motor neurons, and dopaminergic neurons, we performed whole mount immunostaining and visualized different neuronal cell types in vivo. In addition, we assessed neuronal apoptosis, proliferation, oxidation and the integrity of the myelin sheath using TUNEL staining, immunostaining and in situ hybridization. The number of dopaminergic neurons was examined and morphometric analysis was performed to quantify the staining signals for myelin basic protein and apoptosis. RESULTS: We showed that compounds that induce neurotoxicity in humans caused similar neurotoxicity in zebrafish. For example, ethanol induced defects in optic nerves and motor neurons and affected neuronal proliferation; 6-hydroxydopamine caused neuronal oxidation and dopaminergic neuron loss; acrylamide induced demyelination; taxol, neomycin, TCDD and retinoic acid induced neuronal apoptosis. DISCUSSION: Effects of drug treatment on different neurons can easily be visually assessed and quantified in intact animals. These results support the use of zebrafish as a predictive model for assessing neurotoxicity.

Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.

In this research, we optimized parameters for xenotransplanting WM-266-4, a metastatic melanoma cell line, including zebrafish site and stage for transplantation, number of cells, injection method, and zebrafish incubation temperature. Melanoma cells proliferated, migrated and formed masses in vivo. We transplanted two additional cancer cell lines, SW620, a colorectal cancer cell line, and FG CAS/Crk, a pancreatic cancer cell line and these human cancers also formed masses in zebrafish. We also transplanted CCD-1092Sk, a human fibroblast cell line established from normal foreskin and this cell line migrated, but did not proliferate or form masses. We quantified the number of proliferating melanoma and normal skin fibroblasts by dissociating xenotransplant zebrafish, dispensing an aliquot of CM-DiI labeled human cells from each zebrafish onto a hemocytometer slide and then visually counting the number of fluorescently labeled cancer cells. Since zebrafish are transparent until approximately 30 dpf, the interaction of labeled melanoma cells and zebrafish endothelial cells (EC) can be visualized by whole-mount immunochemical staining. After staining with Phy-V, a mouse anti-zebrafish monoclonal antibody (mAb) that specifically labels activated EC and angioblasts, using immunohistology and 2-photon microscopy, we observed activated zebrafish EC embedded in human melanoma cell masses. The zebrafish model offers a rapid efficient approach for assessing human cancer cells at various stages of tumorigenesis.

Zebrafish as a model for developmental neurotoxicity testing.

BACKGROUND: To establish zebrafish as a developmental toxicity model, we used 7 well-characterized compounds to examine several parameters of neurotoxicity during development. METHODS: Embryos were exposed by semistatic immersion from 6 hrs postfertilization (hpf). Teratogenicity was assessed using a modified method previously developed by Phylonix. Dying cells in the brain were assessed by acridine orange staining (these cells are likely to be apoptotic). Motor neurons were assessed by antiacetylated tubulin staining and catecholaminergic neurons were visualized by antityrosine hydroxylase staining. RESULTS: Atrazine, dichlorodiphenyltrichloroethane (DDT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were primarily teratogenic and not specifically neurotoxic. 2,4-dichlorophenoxyacetic acid (2,4-D), dieldrin, and nonylphenol showed specific neurotoxicity; dieldrin and nonylphenol were specifically toxic to catecholaminergic neurons. Malathion, although not teratogenic, showed some nonspecific toxicity. CONCLUSIONS: Teratogenicity measured in 96-hpf zebrafish is predictive of mammalian teratogenicity and is useful in determining whether a compound causes specific neurotoxicity or general developmental toxicity. Induction of apoptosis or necrosis is an indicator of neurotoxicity. An effect on motor neurons in the caudal third of the embryo correlates with expected defects in motility. Overall, our results showed a strong correlation with mammalian data and suggest that zebrafish is a predictive animal model for neurotoxicity screening.

A zebrafish assay for identifying neuroprotectants in vivo.

In this study, we developed an in vivo method to determine drug effects on oxidation-induced apoptosis in the zebrafish brain caused by treatment with L-hydroxyglutaric acid (LGA). We confirmed that LGA-induced apoptosis was caused by oxidation by examining the presence of an oxidative product, nitrotyrosine. Next, we examined the effects of 14 characterized neuroprotectants on LGA-treated zebrafish, including: D-methionine (D-Met), Indole-3-carbinol, deferoxamine (DFO), dihydroxybenzoate (DHB), deprenyl, L-NAME (N(G)-nitro-L-arginine methyl ester), n-acetyl L-cysteine (L-NAC), 2-oxothiazolidine-4-carboxylate (OTC), lipoic acid, minocycline, isatin, cortisone, ascorbic acid and alpha-tocopherol. Eleven of 14 neuroprotectants and 7 of 7 synthetic anti-oxidants exhibit significant protection in zebrafish. Buthionine sulfoximine (BSO), used as a negative control, exhibited no significant protective effects. In addition, three blood-brain barrier (BBB) impermeable compounds exhibited no significant effects. Our results in zebrafish were similar to results reported in mammals supporting the utility of this in vivo method for identifying potential neuroprotective anti-oxidants.

The use of zebrafish for assessing ototoxic and otoprotective agents.

Zebrafish and other fish exhibit hair cells in the lateral-line neuromasts which are structurally and functionally similar to mammalian inner ear hair cells. To facilitate drug screening for ototoxic or otoprotective agents, we report a straightforward, quantitative in vivo assay to determine potential ototoxicity of drug candidates and to screen otoprotective agents in zebrafish larva. In this study, a fluorescent vital dye, DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide), was used to stain zebrafish hair cells in vivo and morphometric analysis was performed to quantify fluorescence intensity and convert images to numerical endpoints. Various therapeutics, including gentamicin, cisplatin, vinblastine sulfate, quinine, and neomycin, which cause ototoxicity in humans, also resulted in hair cell loss in zebrafish. In addition, protection against cisplatin-induced ototoxicity was observed in zebrafish larva co-treated with cisplatin and different antioxidants including, glutathione (GSH), allopurinol (ALO), N-acetyl l-cysteine (l-NAC), 2-oxothiazolidine-4-carboxylate (OTC) and d-methionine (d-MET). Our data indicate that results of ototoxicity and otoprotection in zebrafish correlated with results in humans, supporting use of zebrafish for preliminary drug screening.

Gene expression profile of zebrafish exposed to hypoxia during development.

Understanding how vertebrates respond to hypoxia can have important clinical implications. Fish have evolved the ability to survive long exposure to low oxygen levels. However, little is known about the specific changes in gene expression that result from hypoxia. In this study we used a zebrafish cDNA microarray to examine the expression of >4,500 genes in zebrafish embryos exposed to 24 h of hypoxia during development. We tested the hypotheses that hypoxia changes gene expression profile of the zebrafish embryos and that these changes can be reverted by reexposure to a normoxic (20.8% O(2)) environment. Our data were consistent with both of these hypotheses: indicating that zebrafish embryos undergo adaptive changes in gene expression in response to hypoxia. Our study provides a striking genetic portrait of the zebrafish embryos' adaptive responses to hypoxic stress and demonstrates the utility of the microarray technology as a tool for analyzing complex developmental processes in the zebrafish.

Construction of a zebrafish cDNA microarray: gene expression profiling of the zebrafish during development.

Vertebrate embryogenesis is a complex process controlled by a transcriptional hierarchy that coordinates the action of thousands of genes. To identify and analyze the expression patterns of these genes, we constructed a zebrafish cDNA microarray containing 4512 unique genes identified from zebrafish embryonic heart, adult hearts, and skeletal muscle cDNA libraries. We examined the patterns of gene expression during development in the zebrafish between five time points relative to 12h post-fertilization (hpf). Differentially expressed genes can be grouped into two categories, early genes that are expressed at 5hpf and genes expressed at 48/72/120hpf. Furthermore, we report the utilization of cDNA microarray technology to investigate the adaptive molecular responses of zebrafish to hypoxia during development. Our study provides the first utilization of cDNA microarray in the zebrafish and reveals dynamic changes in levels of gene expression in relation to development and survival of the zebrafish embryos under hypoxic stress.