180 research outputs found
A genome-wide CRISPR screen to generate high-yield cell lines for pandemic influenza vaccine production
All influenza vaccines currently sold in Canada require one fertilized chicken egg to produce roughly one dose of vaccine. During pandemic influenza outbreaks, the limited availability of eggs stresses the ability of this method to deliver vaccine in a timely manner (1). Unlike eggs, cell lines grow exponentially, resulting in virtually limitless substrate for cultivating influenza vaccines. This ability to rapidly scale production during periods of increased demand is ideal for effectively responding to pandemic influenza outbreaks. While promising, cell-based influenza vaccine production suffers from low volumetric yield (~10-fold lower) compared to egg-based methods (2).
In this study, a genome-wide screen was used to identify gene knockouts that increase influenza yield in the HEK-293SF cell line. Viral replication is dependent upon a myriad of cellular factors, for example an estimated 9.5% of human protein-coding genes affect HIV-1 replication (3). Many of these cellular factors are components of the innate immune system and actively inhibit virus replication. Identifying these factors will allow the generation of cell lines where they have been knocked out, enhancing influenza yield.
To carry out the screen, knockouts were induced with a lentivirus-vectored, pooled CRISPR/Cas9 library. Cells were then infected with Green Fluorescence Protein (GFP)-tagged influenza. Cells with a favorable environment for influenza replication expressed high amounts of GFP, allowing them to be collected using Fluorescence Assisted Cell Sorting. Next Generation Sequencing was then used to determine which knockouts enhanced influenza replication.
The results of the screen will inform the generation of high-yield vaccine production cell lines based on the HEK-293SF parent line, advancing efforts towards cell-based vaccine production methods that are able to effectively address pandemic outbreaks. The results also offer insights into the host determinants of influenza infection within the unique environment of bioreactor culture. Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov. 2015;14(3):167-82. Genzel Y. Designing cell lines for viral vaccine production: Where do we stand? Biotechnol J. 2015;10(5):728-40. Bushman FD, Malani N, Fernandes J, D\u27Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, Konig R, Bandyopadhyay S, Ideker T, Goff SP, Krogan NJ, Frankel AD, Young JA, Chanda SK. 2009. Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 5:e1000437
Integrated semi-continuous manufacturing of lentiviral vectors
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Intensified virus production in suspension HEK293SF cells using high inoculum fed-batch or low-perfusion rate cultures with continuous harvest
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292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium
Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application
Intensification of influenza virus production in fed-batch and perfusion cultures of HEK293SF cells
More than half a million people die every year from complications of seasonal influenza, and vaccination stands as the most effective method to prevent and limit outbreaks of the disease. Constant vaccine development based on emerging strains and worldwide distribution of vaccines is a great challenge for public health and vaccine manufacturers, particularly in a potential pandemic scenario. The limited flexibility of the current egg-based production system combined with recent advances in large-scale cell culture techniques have encouraged the development of cell culture processes for influenza vaccine production. While cell culture offers a valuable alternative, productivities are still low when compared to traditional egg-based systems, requiring extensive efforts in process intensification and suspension cell line development.
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Medium- and high-cell-density production of Adeno-Associated virus serotype 6 and the mitigation of cell density effect via medium supplementation
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Characteristics of rVSV-ZEBOV production kinetics in HEK293 and Vero cells
The vesicular stomatitis virus (VSV) can be used as an effective vaccine platform, inducing both cellular and humoral immunity. Because VSV infections of humans are mostly asymptomatic, recombinant VSV (rVSV) can be used as a platform to safely deliver and express foreign antigens. This research study focusses on cell culture production of an rVSV expressing the Ebola virus glycoprotein on its surface (rVSV-ZEBOV). This virus has been demonstrated to be safe to administer to humans. In addition, recent results of a human phase III clinical trial showed that this vaccine can efficiently protect against Ebola virus infection. However, limited data is available in the literature about the growth characteristics of this virus during the production process. In our study, we investigated the influence of multiplicity of infection (MOI), time of infection (TOI), time of harvest (TOH), media components and temperature on the viral titer (TCID50/mL, ddPCR) of rVSV-ZEBOV produced from cell culture. Results are compared between the standard production in the Vero cell line and in a suspension-adapted HEK293-based cell line without serum
Development and validation of a proprietary medium formulation for recombinant subunit vaccines by the Baculovirus Expression Vector System (BEVS)
Insect cells & baculovirus expression vector system (BEVS) is an efficient platform for the production of baculovirus vectors and the expression of recombinant proteins or VLPs (Virus Like Particles). This technology is increasingly used in large-scale manufacturing of human and veterinary vaccines. The production of safe and cost-effective vaccines requires the development of animal origin free and low-cost culture media supporting strong insect cell growth, high yield of baculovirus and sustained expression of recombinant proteins or VLPs.
In this context, Boehringer-Ingelheim decided to develop its own animal origin free media (with similar or better performance than the commercial one) in collaboration with NRC and suppliers. The different steps, feedback and results will be presented in this poster
Proteomic characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles produced in HEK-293SF
One of the major concerns associated with the use of influenza virus-like particles (VLPs) produced in cell culture as vaccine candidates is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out with them not only the viral antigenic proteins but also host cell proteins. In addition, the intrinsic nature of the cells to produce membrane derived vesicles which have similar size to the VLPs and can also contain the antigenic proteins, makes the VLP purification process challenging. Certainly, the expression system and the viral recombinant proteins employed will determine the nature of the proteins within the VLPs. To further characterize cell culture produced-influenza VLPs and contribute to enable their approval as vaccine candidates, the composition and biogenesis of VLPs need to be better understood. In this study we have characterized, by nanoscale liquid chromatography tandem mass spectrometry (n-LC-MS/MS), influenza H1N1 Gag-VLPs produced in human embryonic kidney cells adapted to serum-free medium (HEK-293SF). The cells stably express HA and NA, and the VLPs production occurs following transient transfection with a plasmid containing the gag gene of HIV-1 fused to GFP. Extracellular vesicles (EVs) produced by the unmodified HEK-293SF were also characterized by n-LC-MS/MS. A total of 73 host cell proteins were identified in the VLPs, whereas 98 were detected in the extracellular vesicles. From that, 32 host cell proteins were unique to VLPs while 41 proteins were found in both. Importantly, nucleolin was the most abundant host cell differential protein identified in VLPs while lactotransferrin and heat shock protein 90 were the most present in EVs. This study provides a detailed proteomic description of the VLPs and EVs produced in HEK-293SF as well as a critical discussion of the function of each protein incorporated in both nanoparticles species. The outcome of this research also sheds light on unique target proteins differentially identified either in VLPs and EVs that could potentially be exploited for the development of novel purification protocols to separate EVs from VLPs
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