Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode.

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 106 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4-6.5 × 1013 virions were produced while a control experiment with an ATF system equaled to 1.9 × 1013 virions. For infection at 25 × 106 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 106 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.

Performance of an acoustic settler versus a hollow fiber-based ATF technology for influenza virus production in perfusion.

Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5-3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps. KEY POINTS: • High suspension cell density, immortalized avian cell line, influenza vaccine.

Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production.

Perfusion operation mode remains the preferred platform for production of labile biopharmaceuticals (e.g., blood factors) and is also being increasingly adopted for production of stable products (e.g., monoclonal antibodies). Regardless of the product, process development typically aims at maximizing production capacity. In this work, we investigated the impact of perfusion cultivation conditions on process productivity for production of human factor VIII (FVIII). Recombinant CHO cells were cultivated in bioreactors coupled to inclined settlers and the effects of reducing the temperature to 31°C with or without valeric acid (VA) supplementation were evaluated. Increases in cell specific productivity (qp ) up to 2.4-fold (FVIII concentration) and up to 3.0-fold (FVIII biological activity) were obtained at 31°C with VA compared to the control at 37°C. Biological activity is the most important quality attribute for FVIII and was positively affected by mild hypothermia in combination with the chemical inducer. The low temperature conditions resulted in enhanced product transcript levels, suggesting that the higher qp is related to the increased mRNA levels. Furthermore, a high-producer subclone was evaluated under the perfusion conditions optimized for the parental clone (31°C with VA), yielding increases in qp of 6-fold and 15-fold compared to the parental clone cultivated under the same condition and at 37°C, respectively. The proposed perfusion strategy enables increased product formation without increasing production costs, being potentially applicable to perfusion production of other CHO-derived biopharmaceuticals. To the best of our knowledge, this is the first report showing the benefits of perfusion combining mild hypothermia with VA supplementation.

Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h-1 (doubling time = 32 h). Maximum virus titers in the range of 3.28-3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000-3500 virions/cell and productivities of 0.5-2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1.

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.

Cell therapy modulates expression of Tax1-binding protein 1 and synaptotagmin IV in a model of optic nerve lesion.

PURPOSE: Bone marrow mononuclear cells (BMMCs) have been used with considerable success to improve regeneration and/or functional recovery in animal models of neurologic diseases. Injected into the host, they migrate to the damaged areas and release cytokines and/or trophic factors, which are capable of altering the genetic program of the injured tissue cells. In this study, there was a search for genes with altered expression in a model of optic nerve crush and cell therapy. METHODS: Optic nerve crush was followed by an intravitreous injection of BMMCs or vehicle in adult rats. After 14 days, we obtained a transcriptome screening of the retinas using differential display and automatic sequencing, followed by q-PCR, Western blot, and immunohistochemistry of selected genes and proteins. RESULTS: Among the differentially displayed genes, transcription of the antiapoptotic Tax1-binding protein 1 (Tax1BP1) and Synaptotagmin IV (Syt IV), an immediate early gene, is increased in the treated group. Tax1BP1 expression is robust in the ganglion cell layer and is significantly increased by cell therapy. Syt IV is expressed by activated Müller cells and astrocytes in the retina and optic nerve, without changes in protein levels among the groups. CONCLUSIONS: Tax1BP1 and Syt IV transcription and/or expression are differently modulated by optic nerve crush and BMMC treatment, and might be related to neuronal damage and cell-therapy effects in the retina. The increased expression of Tax1BP1 in the treated eyes could be involved in the neuroprotective effects of BMMCs that were described previously by our group.

Bone marrow mononuclear cells increase retinal ganglion cell survival and axon regeneration in the adult rat.

The central nervous system (CNS) of adult mammals generally does not regenerate, and many studies have attempted to identify factors that could increase neuroprotection and/or axonal outgrowth after CNS lesions. Using the optic nerve crush of rats as a model for CNS injury, we investigated the effect of intravitreal transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) on the survival of retinal ganglion cells (RGC) and on the regeneration of optic axons. Control animals received intravitreal saline injections after lesion. Injections of BMMCs resulted in a 1.6-fold increase in the number of RGCs surviving 14 days after injury. The BMMC-treated animals also had increased numbers of axons, which grew up to 1.5 mm from the crush site, and also had reduced Müller glia activation. Analysis of mRNAs in all conditions revealed an increase in levels of fibroblast growth factor 2 (FGF-2) mRNA in treated animals 14 days after injury. To investigate whether the regenerated axons could reach the brain, we retrograde labeled the RGCs by injecting a lipophilic tracer into the superior colliculus. We also analyzed the expression of NGFI-A in the superficial layers of the superior colliculus as a possible marker of synaptic input from RGC axons. We found evidence that more RGCs were able to reach the brain after treatment and we showed that NGFI-A expression was higher in the treated animals 60 days after injury. These results demonstrate that transplant of BMMCs can increase neuroprotection and neuroregeneration after injury in a model of optic nerve crush, and these effects could be mediated by FGF-2.