A Virus Harvest Unit for the continuous harvesting of lentivirus from suspension cell cultures

For virus particles produced by cell culture systems, free intact virus particles as well as virus particles associated with host cell membranes or other viral particles and damaged virus fragments will be present. To reduce the amount of host cell fragments and/or virus aggregates it is critically important to reduce the shear stress applied to the cell culture during the harvesting step as much as possible. This is mainly accomplished by utilizing a low-shear Virus Harvest Unit (VHUÔ) with large pore size membranes (5-15mM) and by utilizing a low-shear pump. As shown in Table I, the VHUÔ, is able to clarify the broth containing the viral vectors from less than 500 Nephelometric Turbidity Units (NTU) down to less than 16 NTU. In contrast the turbidity of a broth harvested by centrifugation from a batch is around 50-90 NTU and the turbidity of the harvest stream from an acoustic filter (Biosep) is 33 NTU. These results suggest it may be possible to eliminate the secondary clarification steps when using the VHU unit as the virus harvesting process mode. Please click Additional Files below to see the full abstract

HEK293 suspension cell culture platform for production of viruses and viral vectors

Viruses and viral vectors are extensively used as delivery systems for gene and cell therapies, oncotherapies and as vaccines or vectors for display or expression of antigens in vaccination strategies. Over many years, developments in cell culture technologies have been critical to enable mass production of viral vectors and have greatly contributed to facilitate pre-clinical and clinical trials for therapeutic applications. HEK293 is the most popular cell line for the production of adenoviruses (AdV), adeno-associated viruses (AAV), retroviruses and lentiviruses; and HEK293 cell suspension culture is the most efficient system for improved production of r-proteins and viral vectors by large-scale transfection in serum-free media. A HEK293 clone, 293SF-3F6 adapted to grow in suspension in serum-free media, was selected by our institute and banked under GMP conditions. This poster will highlight the current status of various advanced cell culture processes that were developed at our institute over the last 25 years for the production of viruses and viral vectors using HEK293 cell suspension cultures. Through better understanding of nutrimental requirements during the phases of cell growth and virus production, we have developed high yield processes for production of adenoviruses through culture media optimization, fed-batch and perfusion processes. For example, the developed fed-batch process was able to significantly improve the maximum cell density at the virus infection while maintaining specific virus productivity, thus resulting in an 8-fold increase in the yield of adenovirus. A high efficiency transient transfection process was developed for production of lentiviral and AAV vectors. In the case of lentiviral vector, a 100-fold increase of virus yield was obtained through process optimization in a perfusion system. Processes for the production of AdV and AAV have been successfully scaled up in a bioreactor at 500L scale. Processes for the production of other viruses and virus-like particles, such as influenza virus-like particles, will be also presented

Development of a Scalable Process for Lentiviral Vector Mass Production by Transient Transfection

RÉSUMÉ Les vecteurs lentiviraux (LVs) sont considérés comme des véhicules de transfert prometteurs pour des applications en thérapie génique. Cependant, il y a un manque évident de stratégies de production efficaces et transposables à grande échelle. En effet, les techniques actuelles de production ne pourront pas suffire à la génération du nombre de LVs nécessaires pour les évaluations en phases cliniques et éventuellement pour leur commercialisation en cas de succès des essais. Dans ce travail, nous avons tout d’abord fait le point sur les méthodes actuelles de production des LVs et fait ressortir leurs contraintes intrinsèques. Jusqu’à ce jour, la production routinière de LVs se fait presqu’exclusivement à petite échelle avec des cellules adhérentes. Il est clair que ce mode de production ne sera pas suffisant pour répondre aux demandes futures en LVs. Afin de faciliter les essais cliniques et la production de LVs à des fins thérapeutiques après certification par les autorités de règlementation, il apparaît important de mettre au point de nouvelles stratégies de production à grande échelle et permettant l’obtention de vecteurs à hautes concentrations. Une technologie de production de LVs par transfection transitoire de cultures en suspension de la lignée cellulaire HEK293 a été développée. Dans ce procédé transposable à grande échelle, l’opération en mode perfusion a permis de résoudre la problématique associée à la faible stabilité fonctionnelle des LVs. La combinaison de plusieurs approches, incluant la transfection à haute densité cellulaire, la sélection de formulations avancées de milieux de culture et l’ajout de butyrate de sodium en tant qu’additif activateur d’expression, a permis d’augmenter significativement les productivités volumétriques et spécifiques. Ainsi, il a été possible d’augmenter de 100 fois le taux de LVs fonctionnels, que ce soit à petite ou à grande échelle, permettant d’atteindre des titres fonctionnels maximaux en LVs avoisinant les 108 unités de transduction (tu)/mL. Les cinétiques de production ont été évaluées en utilisant plusieurs méthodes de quantification des LVs. Ainsi, on a pu observer que les titres maximaux en LVs fonctionnels sont atteints deux jours après transfection. Ces résultats ont également démontré que la qualité des LVs produits (la qualité étant définie ici comme le rapport entre le nombre de LVs fonctionnels et le nombre de LVs totaux) était généralement faible : de l’ordre de 1 à 4 % du nombre de particules virales----------ABSTRACT Lentiviral vectors (LVs) are promising delivery vehicles for applications in gene therapy. Yet, the field still relies on inefficient and non-scalable production strategies. Current production methods will become a limitation in later clinical evaluation phases and for commercialization when large amounts of LVs need to be generated. In this work, we first reviewed current LV production methods and point out the constraints intrinsic to these protocols. To date, routine LV production is almost exclusively performed in small scale systems using adherent cells. These protocols will not be able to satisfy the anticipated future demands in LVs. For their widespread testing in clinical settings and for the production of LV-based therapeutics after their approval, novel scalable production strategies are needed to robustly produce these vectors at high yield. An optimized protocol for LV production was developed using a HEK293 cell line grown in suspension cultures. In this scalable process, production in perfusion mode addressed the low stability of functional LVs. Several strategies such as transfection at high cell density, selection of advanced medium formulations and addition of the expression-enhancing additive sodium butyrate resulted in significant improvements of volumetric and specific productivity. The overall yield in functional LVs was increased by 100-fold and similar results were obtained for small and bioreactor scale cultures, reaching maximum functional LV titers in the range of 108 transducing units (tu)/mL. The production kinetics under improved conditions was then analyzed employing several LV quantification methods. After transfection, highest functional LV titers were reproducibly found 2 days post-transfection. The results also showed that LV quality, as the ratio of functional to total LV particles was generally low with only 1-4 % of the total viral particles (measured as the number of viral genomes) being functional, i.e. having the ability to transfer genetic information. This ratio was not constant over time when sodium butyrate was added after transfection. LV quality was also increased at higher harvest rates. Our results also indicate that the cytotoxic effects of VSV-G might be limiting for further yield improvements of the current LV production system

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

<p>Abstract</p> <p>Background</p> <p>Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 10⁶cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.</p> <p>Results</p> <p>To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 10⁶cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 10¹¹IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 10¹⁵IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles.</p> <p>Conclusions</p> <p>These results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.</p

Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells

Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Abstract Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL

Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies for rare and more frequent genetic and acquired diseases, as well as in cancer therapies. However, two major challenges need to be overcome in order to generate enough material to treat patients: First, current production platforms result in low titers (stable producer cell lines from adherent cell lines) or are not amenable to large scale production (LV produced by transfection). Next, LVs are known to have a low temperature stability. To address these two challenges, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. Furthermore, productions are performed in perfusion mode in order to operate at high cell densities and address the low LV stability. Please click Additional Files below to see the full abstrac

Development of suspensions adapted Vero cell culture process for production of viruses

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71 and hantaan virus vaccines. Vero cell culture technologies are also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly. Here we report on the adaptation of adherent Vero cells to grow in suspension using a serum-free and animal component-free medium developed in-house. The maximum cell density and cell doubling time of the suspension adapted Vero cells in batch culture grown in the in-house developed medium were similar to or better than what was observed for the adherent Vero cells grown in commercial media. The growth of suspension adapted Vero culture was successfully scaled up to 3 L bioreactor. The Vero cells from various stages (both adherent and adapted) were tested for their authenticity using a Short Tandem Repeat (STR) analysis. The testing result indicates that all Vero cell samples have 100% concordance with the Vero DNA control sample, indicating the suspension adapted cells maintained their genetic stability. Productions of vesicular stomatitis virus (VSV) and influenza virus in adherent culture and suspension adapted culture were compared, showing the suspension adapted Vero cell retained similar viral productivity. The volumetric productivity of VSV in the suspension culture was even higher, and was further increased by almost 200 times when culture was infected at higher cell density and with medium replacement before the virus infection. In contrast, the VSV production decreased when the adherent culture was infected at higher cell density. Additional process development revealed that the maximum cell density in batch culture was doubled, reaching 6x106 cells/mL, when the culture medium was replaced during the process of batch culture, which indicates potential for further increases in product titer

Regulatory T cells with additional COX-2 expression are independent negative prognosticators for vulvar cancer patients

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies