Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics.

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 >100L 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.

Recombinant Protein Production in Large-Scale Agitated Bioreactors Using the Baculovirus Expression Vector System.

The production of recombinant proteins using the baculovirus expression vector system (BEVS) in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantification, bioreactor preparation and operation, the infection process, final harvesting, and primary separation steps for recovery of the product are presented. Furthermore, methods involved with advanced on-line monitoring and bioreactor control, which have a significant impact on the overall process success, are briefly discussed.

Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.

Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.

Monitoring of potential cytotoxic and inhibitory effects of titanium dioxide using on-line and non-invasive cell-based impedance spectroscopy.

Titanium dioxide (TiO2) nanoparticles (NPs) with different sizes and structures were probed for plausible cytotoxicity using electric cell-substrate impedance sensing (ECIS), a non-invasive and on-line procedure for continuous monitoring of cytotoxicity. For insect cells (Spodoptera frugiperda Sf9), the ECIS50 values, i.e., the concentration required to achieve 50% inhibition of the response, differed depending on the size and shape of the TiO2 nanostructure. The lowest ECIS50 value (158 ppm) was observed for the needle shaped rutile TiO2 (10 nm×40 nm, 15.5 nm nominal particle size), followed by 211 ppm for P-25 (34.1 nm, 80% anatase and 20% rutile), 302 ppm for MTI5 (5.9 nm, 99% anatase) and 417 ppm for Hombitan LW-S bulk TiO2 (169.5 nm, 99% anatase). Exposure of TiO2 NPs to UV light at 254 nm or 365 nm exhibited no significant effect on the ECIS50 value due to the aggregation of TiO2 NPs with diminishing photocatalytic activities. Chinese hamster lung fibroblast V79 cells, exhibited no significant cytotoxicity/inhibition up to 400 ppm with P25, MTI5 and bulk TiO2. However, a noticeable inhibitory effect was observed (ECIS50 value of 251 ppm) with rutile TiO2 as cell spreading on the electrode surface was prevented.

Noninvasive cell-based impedance spectroscopy for real-time probing inhibitory effects of graphene derivatives.

Three water-dispersible graphene derivatives, graphene oxide (GO), sulfonated graphene oxide (SGO), and sulfonated graphene (SG), were prepared and probed for their plausible cytotoxicity by non-invasive electric cell-substrate impedance sensing (ECIS). With Spodoptera frugiperda Sf9 insect cells adhered on gold microelectrodes as an active interface, it is feasible to monitor changes in impedance upon exposure to different graphene derivatives. Sf9 insect cells were then exposed to different concentrations of graphene derivatives and their spreading and viability were monitored and quantified by ECIS in real-time. On the basis of the 50% inhibition concentration (ECIS50), none of the graphene derivatives were judged to have any significant cytotoxicity with respect to the chosen cell line as the ECIS50 values were all above 100 µg/mL. However, all graphene derivatives exhibited inhibitory effects on the Sf9 response at the cell spreading level with the following order: SG (ECIS50 = 121 ± 8 µg/mL), SGO (ECIS50 = 151 ± 9 µg/mL), and GO (ECIS50 = 232 ± 27 µg/mL), reflecting differences observed in their ζ-potential and surface area. The presence of phenyl sulfonyl groups in SGO and SG improves their aqueous dispersity which enables these materials to have a greater inhibitory effect on Sf9 insect cells in comparison to GO. Such results were corroborated well with the cell count and viability by the Trypan Blue exclusion assay.

A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis.

BACKGROUND: Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs). METHODS: AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions. RESULTS: PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection. CONCLUSION: The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.

Probing inhibitory effects of nanocrystalline cellulose: inhibition versus surface charge.

NCC derived from different biomass sources was probed for its plausible cytotoxicity by electric cell-substrate impedance sensing (ECIS). Two different cell lines, Spodoptera frugiperda Sf9 insect cells and Chinese hamster lung fibroblast V79, were exposed to NCC and their spreading and viability were monitored and quantified by ECIS. Based on the 50%-inhibition concentration (ECIS(50)), none of the NCC produced was judged to have any significant cytotoxicity on these two cell lines. However, NCC derived from flax exhibited the most pronounced inhibition on Sf9 compared to hemp and cellulose powder. NCCs from flax and hemp pre-treated with pectate lyase were also less inhibitory than NCCs prepared from untreated flax and hemp. Results also suggested a correlation between the inhibitory effect and the carboxylic acid contents on the NCC.

Process optimization and scale-up for production of rabies vaccine live adenovirus vector (AdRG1.3).

Rabies virus is an important causative agent of disease resulting in an acute infection of the nervous system and death. Although curable if treated in a timely manner, rabies remains a serious public health issue in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Control of rabies in wildlife is still an important challenge for governmental authorities. There are a number of rabies vaccines commercially available for control of wildlife rabies infection. However, the vaccines currently distributed to wildlife do not effectively immunize all at-risk species, particularly skunks. A replication competent recombinant adenovirus expressing rabies glycoprotein (AdRG1.3) has shown the most promising results in laboratory trials. The adenovirus vectored vaccine is manufactured using HEK 293 cells. This study describes the successful scale-up of AdRG1.3 adenovirus production from 1 to 500 L and the manufacturing of large quantities of bulk material required for field trials to demonstrate efficacy of this new candidate vaccine. The production process was streamlined by eliminating a medium replacement step prior to infection and the culture titer was increased by over 2 fold through optimization of cell culture medium. These improvements produced a more robust and cost-effective process that facilitates industrialization and commercialization. Over 17,000 L of AdRG1.3 adenovirus cultures were manufactured to support extensive field trials. AdRG1.3 adenovirus is formulated and packaged into baits by Artemis Technologies Inc. using proprietary technology. Field trials of AdRG1.3 rabies vaccine baits have been conducted in several Canadian provinces including Ontario, Quebec and New Brunswick. The results from field trials over the period 2006-2009 demonstrated superiority of the new vaccine over other licensed vaccines in immunizing wild animals that were previously difficult to vaccinate.

Improving adeno-associated vector yield in high density insect cell cultures.

BACKGROUND: Recombinant adeno-associated virus (rAAV) are the most promising vectors for gene therapy. However, large-scale rAAV production remains a challenge for the translation of rAAV-based therapeutic strategies to the clinic. The baculovirus expression vector system (BEVS) has been engineered to produce high rAAV titers in serum-free suspension cultures of insect cells. METHODS: The typical approach of rAAV production in BEVS has been based on a synchronous infection with three baculoviruses at high multiplicity of infection (MOI) [>3 plaque forming units (pfu)/cell]. An alternative approach is to co-infect at low MOI (0.1 pfu/cell). Both strategies (high and low MOI) were compared at a cell density of 1.0 x 10(6) cells/ml in shake-flask experiments. To increase the rAAV titer, a low MOI combined with an initial cell density at infection of 5.0 x 10(6) cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs. RESULTS: An increase of 210% in the rAAV titer (4.7 x 10(11) enhanced transduction units/l) was observed when using low MOI, an effect primarily caused by the increase in cell density. The fed-batch approach resulted in a seven-fold increase of rAAV yield. Controlled operations in bioreactor contributed to further increase the rAAV yield (2.8 x 10(14) vector genomes/l) by 25% in comparison to the shake flask results. CONCLUSIONS: This high yield production process using low MOIs and a feeding strategy successfully addresses several limitations of current rAAV production in insect cells and contributes to position the BEVS system as one of the most efficient for large-scale manufacturing of rAAV vectors.

Probing inhibitory effects of destruxins from Metarhizium anisopliae using insect cell based impedance spectroscopy: inhibition vs chemical structure.

A noninvasive technique based on electric cell-substrate impedance sensing (ECIS) was demonstrated for on-line probing inhibitory effects of five destruxins on Spodoptera frugiperda Sf9 insect cells. Such chemically structurally similar cyclic hexadepsipeptides, were isolated and purified from the fungus Metarhizium anisopliae. Based on a response function, the inhibitory effect of the destruxins was established from determining the half-inhibition concentration (ECIS50), i.e., the level at which 50% inhibition of the cell response was obtained. Probing by cell based impedance spectroscopy indicated that only a slight change in their chemical structures provoked a significant effect on inhibition. Destruxin B was most inhibitory but replacement of a single methyl group with hydrogen (destruxin B2) or addition of a hydroxyl group (destruxin C) significantly reduced the inhibition. The removal of one methyl group and one hydrogen (destruxin A) lowered the inhibitory effect even more whereas the formation of an epoxy ring (destruxin E) in the structure nullified the inhibitory effect.

Probing inhibitory effects of Antrodia camphorata isolates using insect cell-based impedance spectroscopy: inhibition vs chemical structure.

A continuous online technique based on electric cell-substrate impedance sensing (ECIS) was used for probing inhibitory effects on Spodoptera frugiperda Sf9 insect cells exposed to structurally similar compounds isolated and purified from the fruiting bodies of the fungus Antrodia camphorata. Such chemicals consisted of three ergostane-related steroids and five lanosta-related triterpenes, which are known for their diverse properties and use in the formulation of nutraceuticals and functional foods. The half-inhibition concentration (ECIS(50)), the level at which 50% inhibition of the resistance response was obtained, was determined from the response function to establish inhibitory effects of the different isolates. A slight change in their chemical structures resulted in significant effects on inhibition as probed by impedance spectroscopy. The ergostane-related steroids were mostly inhibitory, but replacing their ketone groups with hydrogen or hydroxyl groups significantly reduced the inhibition. Similarly, the addition of methyl or carboxymethyl groups also lowered the inhibition. Removal of the double bond conjugation within the rings (sulfurenic acid) of the isolate drastically reduced the inhibition.

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system.

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.