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3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future.

Since the early 2000’, regulation agencies have encouraged viral vaccine manufacturers to implement in-process and real-time monitoring tools in production processes. Even if more assays have been recently developed, none of the novel viral particle quantification technologies can monitor virus levels and their secretion kinetics within production vessels. Vaccine manufacturers still rely on offline cell-based infectivity assays and antigen amount quantification to monitor their processes. The present study describes the development of the first automated biosensor for at-line monitoring of influenza virus production. It involves coupling a fetuin-based SPRi quantitative biosensor with an automated sampler of culture broth and a consecutive clarification setup via an acoustic filter. The SPRi response of different viral strains produced in two distinct cell production platforms was qualified. We demonstrated that fetuin-based quantitative SPRi is a robust, potency-indicating, and universal analytical technology for quantifying bioactive influenza virus particles. It was validated with both purified and complex matrices. Finally, an influenza viral production kinetic was monitoredonlinefor three days. This novel online tool enabled the access in real-time to total bioactive viral particles from early production phases (8hpi).

In recent years, numerous strategies have emerged to answer the growing demand for graftable tissues. Tissue engineering andin-vitroproduction are one of them. Among all the engineered tissues, skin is one of the most advanced. Nevertheless, biofabrication of graftable and fully functional skin substitutes is still far from being reached. Skin reconstruction, particularly dermis, necessitates cultivation and maturation for several weeks (> 3 weeks) to recover the tissue’s composition and functions, which prevent its transfer to clinical applications. Thus, several strategies, including 3D bioprinting, have been explored to accelerate these productions. In the present study, based on the successful application of 3D bioprinting achieved by our group for skin reconstruction in 21 days, we propose to detail the biological behaviors and maturation phases occurring in the bioprinted skin construct thanks to a descriptive approach transferred from the bioprocess field. The aim is to comprehensively characterize dermis construct maturation phases (cell proliferation and ECM secretion) to master later the interdependent and consecutive mechanisms involved inin-vitroproduction. Thus, standardized quantitative techniques were deployed to describe 3D bioprinted dermis proliferation and maturation phases. Then, in a second step, various parameters potentially impacting the dermis reconstruction phases were evaluated to challenge our methodology and reveal the biological behavior described (fibroblast proliferation and migration, cell death, ECM remodeling with MMP secretion). The parameters studied concern the bioprinting practice including various printed geometries, bioink formulations and cellular physiology in relation with their nutritional supplementation with selected medium additives.

Contraction assay based on surface measurement have been widely used to evaluate cell contractility in 3D models. This method is straightforward and requires no specific equipment, but it does not provide quantitative data about contraction forces generated by cells. We expanded this method with a new biomechanical model, based on the work-energy theorem, to provide non-destructive longitudinal monitoring of contraction forces generated by cells in 3D. We applied this method on hydrogels seeded with either fibroblasts or osteoblasts. Hydrogel mechanical characteristics were modulated to enhance (condition HCAHigh: hydrogel contraction assay high contraction) or limit (condition HCALow: hydrogel contraction assay low contraction) cell contractile behaviors. Macroscopic measures were further correlated with cell contractile behavior and descriptive analysis of their physiology in response to different mechanical environments. Fibroblasts and osteoblasts contracted their matrix up to 47% and 77% respectively. Contraction pressure peaked at day 5 with 1.1 10-14 Pa for fibroblasts and 3.5 10-14 Pa for osteoblasts, which correlated with cell attachment and spreading. Negligible contraction was seen in HCALow. Both fibroblasts and osteoblasts expressed α-SMA contractile fibers in HCAHigh and HCALow. Failure to contract HCALow was attributed to increased cross-linking and resistance to proteolytic degradation of the hydrogel.

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.

Large bioprinted tissues (dm3) are more and more accessible but their in vitro culture and maturation conditions stay an unchartered territory. In the present report, we aim to present a preliminary study of endothelialized large bioprinted tissues (fibroblast and human dermal microvascular endothelial cells) maturation using silicone 3D printed perfusion system (bioreactor). Computational Fluid Dynamics (CFD) simulation was used to relate the theoretical culture medium flow path within the large bioprinted tissue with the actual tissue morphology and composition, obtained through histological observations. The obtained results demonstrate the positive impact of using dynamic maturation conditions (300 mL/h culture medium flow rate) on the extracellular matrix production and the conservation of the large bioprinted tissue internal geometry. Clear differences between static and dynamic culture conditions were herein found. Finally, typical microvascular organization, composed of human dermal microvascular endothelial cells organized around an open lumen, were found within the large bioprinted tissue.

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (3%) and a greater analytical range (0.03-20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID's one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

International audience; Over the last decade industrial manufacturing of viral vaccines and viral vectors for prophylactic and therapeutic applications is experiencing a remarkable growth. Currently, the quality attributes of viral derived products are assessed only at the end-point of the production process, essentially because in-process monitoring tools are not available or not implemented at industrial scale. However, to demonstrate process reproducibility and robustness, manufacturers are strongly advised by regulatory agencies to adopt more on-line process monitoring and control. Dielectric spectroscopy has been successfully used as an excellent indicator of the cell culture state in mammalian and yeast cell systems. We previously reported the use of this technique for monitoring influenza and lentiviral productions in HEK293 cell cultures. For both viruses, multi-frequency capacitance measurements allowed not only the on-line monitoring of the production kinetics, but also the identification of the viral release time from the cells. The present study demonstrates that the same approach can be successfully exploited for the on-line monitoring of different enveloped and non-enveloped virus production kinetics in cell culture processes. The on-line monitoring multi-frequency capacitance method was assessed in human HEK293 and Sf9 insect cells expression systems, with viral productions initiated by either infection or transfection. The comparative analyses of all the data acquired indicate that the characteristic capacitance signals were highly correlated with the occurrence of viral replication phases. Furthermore the evolution of the cell dielectric properties (intracellular conductivity and membrane capacitance) were indicative of each main replication steps. In conclusion, multi-frequency capacitance has a great potential for on-line monitoring, supervision and control of viral vector production in cell culture processes.

Introduction: Influenza Virus-like Particles (VLPs) are one of the most promising vaccine strategies to complement traditional egg-based processes and contribute to shortening the response time when facing future pandemics. Research programs have taken advantage of the potential of this approach to produce influenza VLPs on a variety of cellular platforms, reaching the industrial level of development and recent commercialization. Area covered: This review aims to give an overview of available strategies for influenza-VLP production and their respective stages of development, from small-scale preclinical studies to large-scale industrial processes. Recent trends and fulfillments in purification schemes of influenza VLP were also reviewed with regards to quality and potency requirements that go along with influenza vaccine manufacturing. Expert opinion: In the next five years, it is expected that there will be licensing of new influenza vaccine products based on VLP strategy. Few VLP upstream processes are mature enough and close to fully complement or seriously concurrence the ovoculture process. Nevertheless, many improvements have yet to be achieved in downstream processes. In the next few years, research efforts in this field are expected to provide purification strategies and tools to achieve higher recovery yields and improve the cost-effectiveness of VLP processes.

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris-HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 10(9) and 1 × 10(11) virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 10(8) and 4.35 × 10(9) whereas the limit of quantification was between 6.90 × 10(8) and 1.45 × 10(10)VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.

International audience; Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015–2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required tomeet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains.

International audience; Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at \textgreater100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×106cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9.05 log TCID50/ml for A/Panama/2007/99 influenza H3N2. Porcine strains were also greatly rescued with titres from 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, viral kinetics showed maximal titers reached at 24h post-infection for most of the strains, allowing early harvest time (Time Of Harvest: TOH). The B strains present specific production kinetics with a delay of 24h before reaching the maximal viral particle release. Process optimization on H1N1 2009 human pandemic strain allowed identifying best operating conditions for production (MOI, trypsin concentration, cell density at infection) allowing improving the production level by 2 log. Our results suggest that the DuckCelt®-T17 cell line is a very promising platform for industrial production of influenza viruses and particularly for avian viral strains.

The following article is a report from a workshop on Quality-by-Design (QbD) held at the 7th European Symposium on Biochemical Engineering Science (7 September 2008, Faro, Portugal).The aim of the workshop was to provide an update on the present status of using QbD in biotechnology-related applications in the pharmaceutical industry. The report summarizes the essential parts of the presentations and covers the industrial, academic, and regulatory aspects of QbD. It concludes with recommendations for further work and development.

Background Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus’ fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen’s eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. Methods In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. Results For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. Conclusions This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0152-x) contains supplementary material, which is available to authorized users.

Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals including viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of recombinant proteins and a well-established system for the production of viral vectors. Understanding metabolic requirements for high productivity in HEK293 cells remains an important area of investigation. Many authors have presented approaches for increased productivity through optimization of cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed to improving feeding strategies by addition of exhausted or critical substrates and eventually removal of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for optimization through better understanding of the cellular metabolism under different operating conditions. This review will present both approaches and their successes with regards to improvement of viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating remaining issues to address and problems to resolve to maximize the productivity of viral vectors in HEK293 cells.

Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.

International audience; This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 10(6) cell mL(-1). Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f(c), which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.

The Vero cell line is extensively used in the manufacturing of human and veterinary vaccines. For security and quality reasons, it was recently adapted to animal component-free culture conditions. But, this adaptation pointed out a lack of understanding of the Vero cell physiology and metabolism. In fact, very few studies deal with Vero cell culture in serum-free media, and they are often focused on virus production. Thus, more efforts should be made to better understand the Vero cell metabolism in absence of serum for optimization of industrial processes. In this work, we studied the central Vero cell metabolism based on the main cell nutrients such as glucose, glutamine and other amino acids. Metabolic studies were performed with extracellular and intracellular analyses all over cultures. Various operating conditions were evaluated by substituting glucose or glutamine with fructose, asparagine, glutamate, glutamax or pyruvate. Kinetic parameters were calculated and compared. Based on the results, a metabolic flux scheme was proposed to represent Vero cell metabolism in serum-free medium.

Based on the regulation agency requirements, one of the main challenges for biological production is to improve in-situ monitoring of animal cell industrial processes (PAT approach). Further than pH, pO2 and cell density which are already on-line monitored, the medium component concentrations are key information to improve the process efficiency and ensure the final product quality. Near infrared spectroscopy (NIRS) was proved to be a good candidate to in-situ monitor these parameters in biochemical processes. Nevertheless, only few works reported NIRS use for animal cell cultures and they were only focused on single suspension cells. Consequently, our study was interested in proposing NIRS as a new in-situ tool to quantify glucose and lactate levels, in the particular case of adherent Vero cells highly used in vaccine production. The NIRS quantification technique was challenged with the implementation of various operating conditions close to the industrial process, such as punctual exchange of culture medium with cells grown on microcarriers in stirred bioreactors.

Background 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 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products. Results 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 × 106 cells/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 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 IVP 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. Conclusions 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.

International audience; In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l−1, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l−1 for glucose and 0.29 g l−1 for lactate.

International audience; A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes.

Background Cell-culture based vaccines are a valuable alternative to egg-produced vaccines. The equivalent of 1500 influenza vaccine doses can be produced in a 1L bioreactor within 48 hours with HEK293 cells. The kinetics of production of H1N1 A/Puerto Rico/8/68 in HEK293 cells has been previously characterized by our group [1]; higher viral production is achieved when cells are infected at a multiplicity of infection (MOI) of 0.01 compared to an MOI of 1. Also, two cycles of infection take place: a first round of virus is produced at 8 hours post-infection (hpi) which then infect cells again and lead to a second viral exit at 16 hpi. Infection triggers a cascade of intracellular responses which contributes to viral replication, but eventually leads to cell death. Most studies focus on one pathway at a time, and aim at limiting the extent of the infection through the use of inhibitors. Our goal however is to increase viral yield and quality. The main objective is therefore to understand and manipulate the molecular events taking place at the cellular level upon influenza infection and replication, with particular attention to key time points corresponding to viral production and exit. Signaling pathways examined include the Akt, mTOR and ERK pathway, and their activation levels were determined by measuring their phosphorylation states.

Background In cell-based processes, and particularly in viral vaccine production, cell growth and death are strategic informations to obtain for process monitoring (ie. scale-up, determination of MOI, TOI, and harvest time). Dielectric spectroscopy is a tool which was increasingly implemented on cell-culture bioreactors as it presents great potentials, compared to other methods, for the in-line monitoring of these two crucial parameters. Considering viral vaccine production, Vero cells are one of the most employed cell platform. But, due to its adherent characteristics, few in-line techniques were developed for cell density monitoring, on the contrary to the ones available for suspension cells. In addition, it should be underline that no in-line technique exists for quantification or detection of the mammalian cell death, despite the importance of this parameter for cell culture processes.

Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. However, while cell culture allows for more versatility regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. FDA’s Process analytical Technology (PAT) and Quality by Design (QbD) guidances impels vaccine manufacturers to implement on-line technologies to monitor both critical process parameters (CPP) and the bioproduct’s critical quality attributes (CQA) within bioprocesses. Today, Influenza vaccine potency is evaluated by single-radial immunodiffusion (SRID), which requires the annual production of strain-specific reagents that constitutes a potential bottleneck for the release of vaccine lots and cannot be implemented for in-process monitoring. On the other hand, while recent physical quantification techniques appear more relevant than traditional infectivity assays for influenza virus monitoring as the ratio of total to infectious influenza virus vary from 10-1000, they are not potency-indicating. During this thesis, we developed and evaluated a potency-indicating assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigens and whole influenza viruses based on quantitative surface plasmon resonance imaging (SPRi). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses. Influenza vaccine and virus samples derived from various cell culture platforms were processed onto the sensor to evaluate viral production kinetics. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng HA/mL-20 μg HA/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, as well as a better reproducibility than SRID and its potential alternatives (