Gene Editing/Gene Therapies: BIOREACTOR RECOMBINANT PRODUCTION OF SARS-COV-2 VIRUS ANTIGENS IN CHO CELL CLONES BY USING NEW UCOE LENTIVIRAL VECTOR SYSTEM

Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy

Anticoagulant effect of Feijoa sellowiana extracts generated by different biotechnological techniques.

Blood clotting has become one of the most dangerous side effects associated with Corona virus, as well as the high level of cholesterol and triglycerides in the blood. Therefore, it has become necessary to use medicinal plants that are biologically safe and containing anti-clotting compound. Feijoa sellowiana represents a prolific source diverse compounds that may have thrombolytic activity. Therefore, the main research point is the production and scaling up of a target contents that have anticoagulants by using biotechnological techniques; calli production, and bioreactors and assessed their activity through in-vivo study. Murashige and Skoog (MS) medium enriched with varying concentrations of benzyl adenine (BA) and naphthalene acetic acid (NAA) was used to cultivate calli and cell suspension cultures from F. sellowiana seeds. Bioreactors were employed to boost active constituent's production. Moreover, the bioreactor physical factors such as effect of controlled or uncontrolled pH medium were investigated. The leaves of the main plant were extracted by ethanol 70% and polar and non-polar extracts were also prepared. The ethanol extract of calli and cells resulting from bioreactors were also prepared. All prepared extracts were subjected to chemical analysis by HPLC, in-vitro antioxidant assays, in-vivo anticoagulant activity and histopathological examination. Calli and cell suspension cultures were produced by using MS medium fortified with 1 mg/L BA+ 0.1 mg/L NAA. It was found that culturing of cell cultures in a bioreactor with uncontrolled pH and aeration at the value of 0.5 L/min gave the maximum and economical fresh and dry weights of the plants. After evaluation of all extracts; it was found that the calli ethanol extract for each plant was the highest value of total phenolic and total flavonoid contents either quantitatively or qualitatively. All extracts of Feijoa had antioxidant activity. The IC50 of the DPPH of Feijoa calli extract was 13.45 µg/mL, it was also confirmed by FRAP and ABTs values. Feijoa calli extract decreased platelet aggregation by suppression of thrombin, extended aPTT, PT, bleeding and clotting times. It was safer than warfarin medication. From these findings the authors can conclude that Feijoa had highly anticoagulant activity and the calli production achieved the goal of the enhancement of the phenolic constituent and thus their activity.

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing.

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 µg/mL and 13.4 µg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.

Recent advances in optimization strategies and applications of plant bioreactors

Plant bioreactors have been the central part of molecular pharming. Vaccines, antibodies and functional foods produced by plant bioreactors with the benefits of cost-effectiveness, high scalability, rapid production, enabling post-translational modification, and no harmful pathogens contamination are increasingly accepted by the public. In February 2022, Health Canada approved the world's first plant-derived human vaccine Covifenz® for the prevention and treatment of COVID-19, marking the advent of the era of molecular pharming represented by plant bioreactors. This paper elaborates the development history of plant bioreactors, with the main host species representatives of leafy plants and seed plants, the stable and transient expression systems construction for various applications, as well as the enhancement strategies through promoter and codon optimization, "humanization” of glycosylation process, inhibition of gene silencing and protease activity, and also summarize the application of plant-derived protein products, which aim to provide a theoretical and application basis for the development of plant bioreactors. © 2023, China Biotechnology Press. All rights reserved.

Current pandemic COVID-19 vaccine strategies and development: a comprehensive review

The coronavirus disease-19 pandemic with the characteristics of asymptomatic condition, long incubation period and poor treatment has influenced the entire globe. Coronaviruses are important emergent pathogens, specifically, the recently emerged sever acute respiratory syndrome coronavirus 2, the causative virus of the current COVID-19 pandemic. To mitigate the virus and curtail the infection risk, vaccines are the most hopeful solution. The protein structure and genome sequence of SARS-CoV-2 were processed and provided in record time;providing feasibility to the development of COVID-19 vaccines. In an unprecedented scientific and technological effort, vaccines against SARS-CoV-2 have been developed in less than one year. This review addresses the approaches adopted for SARS-CoV-2 vaccine development and the effectiveness of the currently approved vaccines.Copyright © 2023, Finlay Ediciones. All rights reserved.

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.

Application of matrices for the development of next-gen bioreactors from COVID-19 waste management prospects

Anthropogenic activities have severely altered the innate state of the environment and the damage incurred had manifested itself as a catastrophe. One of the best examples is the recent occurrence of the COVID-19 pandemic. Also, a significant increase in the presence of pathogenic viruses including the novel coronavirus in the wastewater due to medical discharge is an alarming concern. COVID-19 was almost inevitable a year ago, but the rigorous efforts of the scientific community made it possible. Incorporation of several tools of biotechnology was made for diagnosis, for continuous mass production of therapeutics and vaccines, viral genome editing, etc. to combat the virus. The bioreactor is one such tool;it forms the core of biological processes and is constantly emerging as a powerful tool in several applications namely biopharmaceuticals, bioenergy production, industrial products synthesis, wastewater treatment, solid waste management, etc. Matrix is an integral part of bioreactor providing conducive environment for the occurrence of biological process. This study highlights the recent progress in the novel design and applications of bioreactors from the perspective of the matrix. The facets on how bioreactors can help in post-covid waste management are also discussed. © 2022 Elsevier Inc. All rights reserved.

[Clinical applications of iPS cell-derived platelets].

The COVID-19 pandemic has cast a shadow over transfusion medicine based on the blood donation system. However, managing alloimmune platelet transfusion refractoriness (allo-PTR) has already been difficult. As a first step toward resolving this issue using induced pluripotent stem cell-derived platelet products (iPSC-PLTs), a clinical trial of autologous products (iPLAT1) was conducted in a patient with allo-PTR caused by anti-HPA-1a antibodies who had no compatible donor, and safety was confirmed. To produce iPSC-PLTs, a master cell bank (MCB) of expandable megakaryocyte lines (imMKCLs) is established from iPSCs. From this MCB, iPSC-PLTs are manufactured using a newly developed turbulent-type bioreactor and various compounds. Their quality, safety, and efficacy are confirmed by extensive preclinical studies. Based on the findings of the iPLAT1 study, a clinical trial of allo-transfusion of HLA homozygous iPSC-PLTs is currently ongoing and HLA class I-deficient O-type universal iPSC-PLTs are also being developed. iPSC-PLTs are expected to solve various problems, including allo-PTR in platelet transfusion, and greatly contribute to the advancement of transfusion medicine.

Gram‐Scale mRNA Production Using a 250‐mL Single‐Use Bioreactor

The COVID‐19 pandemic triggered an unprecedented surge in development of mRNA‐based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single‐use bioreactors are traditionally used for cell‐based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell‐free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed‐batch IVT approach, and demonstrate the feasibility of continuous fed‐batch production, paving the way towards continuous manufacturing of mRNA. [ FROM AUTHOR] Copyright of Chemie Ingenieur Technik (CIT) is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Molecular farming using transgenic rice endosperm.

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.

Removal performance of SARS-CoV-2 in wastewater treatment by membrane bioreactor, anaerobic-anoxic-oxic, and conventional activated sludge processes.

The potential risk of SARS-CoV-2 in treated effluent from a wastewater treatment plant (WWTP) is concerned since SARS-CoV-2 is contained in wastewater during the COVID-19 outbreak. However, the removal of SARS-CoV-2 in WWTP has not been well investigated. The objectives of this study were (i) to clarify the removal performance of SARS-CoV-2 during wastewater treatment, (ii) to compare the removal performance of different secondary treatment processes, and (iii) to evaluate applicability of pepper mild mottle of virus (PMMoV) as a performance indicator for the reduction of SARS-CoV-2 RNA in wastewater treatment. Influent wastewater, secondary-treatment effluent (before chlorination), and final effluent (after chlorination) samples were collected from a WWTP from May 28 to September 24, 2020, during the COVID-19 outbreak in Japan. The target WWTP had three parallel treatment systems employing conventional activated sludge (CAS), anaerobic-anoxic -oxic (A2O), and membrane bioreactor (MBR) processes. SARS-CoV-2 in both the liquid and solid fractions of the influent wastewater was concentrated and quantified using RT-qPCR. SARS-CoV-2 in treated effluent was concentrated from 10 L samples to achieve a detection limit as low as 10 copies/L. The log reduction value (LRV) of SARS-CoV-2 was 2.7 ± 0.86 log10 in CAS, 1.6 ± 0.50 log10 in A2O, and 3.6 ± 0.62 log10 in MBR. The lowest LRV observed during the sampling period was 2.8 log10 in MBR, 1.2 log10 in CAS, and 1.0 log10 in A2O process, indicating that the MBR had the most stable reduction performance. PMMoV was found to be a good indicator virus to evaluate reduction performance of SARS-CoV-2 independent of the process configuration because the LRV of PMMoV was significantly lower than that of SARS-CoV-2 in the CAS, A2O and MBR processes.

Process Development and Manufacturing: A SUPPLY CHAIN CRISIS STORY: CULTURE BAG SHORTAGE ENFORCED VALIDATION OF AN ALTERNATIVE EXPANSION SYSTEM FOR CAR T CELLS

Background & Aim: The recent supply chain crisis highlights a need to establish alternative manufacturing (MFG) protocols ensuring continuity of existing and new cell therapy (CT) clinical trials. Our academic CT program, and likely others, experienced purchasing delays and restrictions caused by diversion of critical supplies to meet COVID-19- related research demands and/or reduced vendor capacity due to resource constraints, including attrition of skilled workforce. Mitigation strategies aimed at creating process redundancies overcome production challenges resulting from a scarcity of goods. Here, we validated an alternative ex vivo culture system to clinically MFG lentiviral vector (LV) modified CAR T cells due to limited availability of cell expansion culture bags for the Wave bioreactor, a critical unit of operation that we have used to successfully MFG thousands of gene-modified T cell products for 30+ clinical trials. Methods, Results & Conclusion: The disposable G-REX culture vessels were compatible and seamlessly integrated with our closed system platform. Mesothelin CAR T cells were manufactured in parallel via the G-REX or conventional Wave bioreactor using consented patient starting material. Critical quality attributes of the final T cell products, including viability, transduction efficiency, phenotype and function were assessed. Transduction efficiencies assessed by flow cytometry and/or molecular qPCR were lower in products generated in the G-REX compared to the wave using the same multiplicity of infection. However, at least 50-fold expansion was achieved, with cell viabilities greater than 90% and with comparable cellular phenotypes. The Meso CAR T cells generated by either process were capable of eliciting CAR-mediated cytotoxicity and effector cytokine production. Strikingly, 2-4 billion T cells were harvested from a starting seed number of just 50 million T cells in the 1L G-REX, which may be sufficient to meet most protocol- specified cell therapy doses, suggesting that a full apheresis collection may not be needed. Notably, this process required just 1/3 of the starting material, 1/5 of the media and decreased manual effort through culture duration compared to the Wave. Additionally, the reduced reliance on specialized capital equipment combined with a small footprint enables simultaneous MFG of several immunotherapy products. These advantages propose consideration in replacement of current expansion platform as well as validating an alternative process for MFG CAR T cells.

iPSC: SELECTION OF O-VE IPSC CLONES FOR HIGH-DENSITY RED BLOOD CELL PRODUCTION IN A SCALABLE PERFUSION BIOREACTOR SYSTEM

Background & Aim: Blood is one of the most vital resources in modern medicine. Blood transfusions have become an essential and often lifesaving procedure for accidents, during surgery, for patients with chronic disorders such as anemia, sickle cell disease, cancer, and myriad other circumstances. However, despite the rapidly growing world population, the availability of healthy blood donors is declining with aging populations. Furthermore, natural and man- made calamities often produce sudden and concentrated shocks in demand, which strains global supply chains. The COVID-19 pandemic has demonstrated this issue on a global scale by reducing the number of blood drives and donations, resulting in 39% of blood centers in the United States being left with only one- to two-day supplies, and a 50% drop of blood units collected in countries such as Zambia. Additionally, storage limitations of 42 days for donor blood limits stock availability during peak demand. Large-scale generation of universal red blood cells (RBCs) from O-ve human induced pluripotent stem cells (hiPSCs) offers the potential to alleviate blood shortages and provide a secure year-round supply. Mature iPSC-derived RBCs and reticulocytes could also find important applications in research in malaria and COVID-19 studies. (Figure Presented) Fig. 1 ( 700). Methods, Results & Conclusion: In this study, we have reprogrammed hiPSC from CD34+ O-ve cells and demonstrated the smallscale generation of high-density cultures of erythroblasts in a stirred perfusion bioreactor system. Twenty O-ve iPSC lines were derived, screened, and characterized for their ability to differentiate towards the erythroid lineage, showing high expression of mesoderm (KDR+, 64.9%), hematopoietic (CD34+/CD45+, 68.4%;CD34+/CD43+, 84.9%), and erythroid markers (CD235a+, 83,5%), and were able to undergo enucleation in vitro. Using the best clones, we were able to achieve erythroblast peak cell density of 34.7 million cells/mL with 92.2% viability in an Applikon perfusion bioreactor using an ultrasound system (Sonosep) to concentrate cells while removing waste media. This resulted in a cumulative-fold expansion of over 1,500 after 29 days of culture. Cells carried O2 effectively as demonstrated by hemoglobin dissociation curves. The perfusion culture platform paves the way for controlled high-density bioreactor culture for the generation of RBCs.

Development and Scalable Production of Newcastle Disease Virus-Vectored Vaccines for Human and Veterinary Use.

The COVID-19 pandemic has highlighted the need for efficient vaccine platforms that can rapidly be developed and manufactured on a large scale to immunize the population against emerging viruses. Viral-vectored vaccines are prominent vaccine platforms that have been approved for use against the Ebola virus and SARS-CoV-2. The Newcastle Disease Virus is a promising viral vector, as an avian paramyxovirus that infects poultry but is safe for use in humans and other animals. NDV has been extensively studied not only as an oncolytic virus but also a vector for human and veterinary vaccines, with currently ongoing clinical trials for use against SARS-CoV-2. However, there is a gap in NDV research when it comes to process development and scalable manufacturing, which are critical for future approved vaccines. In this review, we summarize the advantages of NDV as a viral vector, describe the steps and limitations to generating recombinant NDV constructs, review the advances in human and veterinary vaccine candidates in pre-clinical and clinical tests, and elaborate on production in embryonated chicken eggs and cell culture. Mainly, we discuss the existing data on NDV propagation from a process development perspective and provide prospects for the next steps necessary to potentially achieve large-scale NDV-vectored vaccine manufacturing.

Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD.

Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.

Bioengineering Outlook on Cultivated Meat Production.

Cultured meat (also referred to as cultivated meat or cell-based meat)-CM-is fabricated through the process of cellular agriculture (CA), which entails application of bioengineering, i.e., tissue engineering (TE) principles to the production of food. The main TE principles include usage of cells, grown in a controlled environment provided by bioreactors and cultivation media supplemented with growth factors and other needed nutrients and signaling molecules, and seeded onto the immobilization elements-microcarriers and scaffolds that provide the adhesion surfaces necessary for anchor-dependent cells and offer 3D organization for multiple cell types. Theoretically, many solutions from regenerative medicine and biomedical engineering can be applied in CM-TE, i.e., CA. However, in practice, there are a number of specificities regarding fabrication of a CM product that needs to fulfill not only the majority of functional criteria of muscle and fat TE, but also has to possess the sensory and nutritional qualities of a traditional food component, i.e., the meat it aims to replace. This is the reason that bioengineering aimed at CM production needs to be regarded as a specific scientific discipline of a multidisciplinary nature, integrating principles from biomedical engineering as well as from food manufacturing, design and development, i.e., food engineering. An important requirement is also the need to use as little as possible of animal-derived components in the whole CM bioprocess. In this review, we aim to present the current knowledge on different bioengineering aspects, pertinent to different current scientific disciplines but all relevant for CM engineering, relevant for muscle TE, including different cell sources, bioreactor types, media requirements, bioprocess monitoring and kinetics and their modifications for use in CA, all in view of their potential for efficient CM bioprocess scale-up. We believe such a review will offer a good overview of different bioengineering strategies for CM production and will be useful to a range of interested stakeholders, from students just entering the CA field to experienced researchers looking for the latest innovations in the field.

High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor.

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.

Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures.

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.

Advanced Applications for Protein and Compounds from Microalgae.

Algal species still show unrevealed and unexplored potentiality for the identification of new compounds. Photosynthetic organisms represent a valuable resource to exploit and sustain the urgent need of sustainable and green technologies. Particularly, unconventional organisms from extreme environments could hide properties to be employed in a wide range of biotechnology applications, due to their peculiar alleles, proteins, and molecules. In this review we report a detailed dissection about the latest and advanced applications of protein derived from algae. Furthermore, the innovative use of modified algae as bio-reactors to generate proteins or bioactive compounds was discussed. The latest progress about pharmaceutical applications, including the possibility to obtain drugs to counteract virus (as SARS-CoV-2) were also examined. The last paragraph will survey recent cases of the utilization of extremophiles as bio-factories for specific protein and molecule production.