A comparison of orbitally-shaken and stirred-tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred-tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5L OSR and a 3L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH-controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process-dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension-adapted animal cells. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174-1180, 2016

Transient Gene Expression in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

The growing demand of biopharmaceutical products is boosting the market for therapeutic recombinant proteins (r-proteins). More than half of the 140 r-proteins that have gained approval for human therapeutic use are manufactured in mammalian cells that make possible complex post-translational modifications, like glycosylation. In the industry, r-proteins are manufactured by stable gene expression (SGE) following Good Manufacturing Practice (GMP) standards. Transient gene expression (TGE) in mammalian cells is an alternative method for the expression of r-proteins whose main advantages are rapidity and flexibility. TGE involves the expression of the protein of interest from plasmids, which are transfected into the cells with a non-viral DNA transfecting agent, usually polyethylenimine (PEI). DNA is transcribed from the plasmids without the latter having been integrated into the host genome. Despite the recent improvements in r-protein titers by TGE and the scale up of this method to the 100-L scale, TGE remains so far a tool for protein production for research purposes. Therefore, to determine if TGE can be used for manufacturing therapeutic r-protein following GMP standards, different features related to reproducibility and product quality from TGE of a recombinant anti-rhesus D immunoglobulin G in HEK-293E cells using linear PEI were characterized. To test the effects of the size distribution and chemical structure of PEI on TGE, different commercially available PEIs were characterized and their impact on transfection and r-protein expression were assessed. The results showed that both the molecular weight distribution and the chemical structure of the PEI had an effect on TGE. However, the small variations in size distribution and structure between the batches of PEI from the same supplier did not affect significantly the transfection and r-protein production by TGE. Then, the fate of PEI and plasmid DNA in cell culture over time and their removal from the final r-protein during purification were determined and measured. Most of the PEI and pDNA remained in the cell culture medium after transfection. However, both PEI and plasmid DNA, which are additional components in TGE compared to processes based on recombinant cell lines, could be reduced to a concentration below the limit of detection of the assays after Protein A affinity purification of the antibody. To test the reproducibility of a TGE process, 10 batches of transfected cells were run in 500-mL orbitally shaken bottles. The cell specific productivity of the antibody was 20.2 ± 2.6 pg.cell-1.day-1 on average for the 10 batches. The purity and size consistency of the recombinant antibody produced in those 10 batches was demonstrated by gel electrophoresis and size exclusion chromatography. Then, the glycosylation profile of the antibody produced by TGE in HEK-293E cells was determined by mass spectrometry and was reproducible over the 10 batches. Moreover, it was similar to the glycosylation profile of the antibody produced by 3 stable HEK-293E cell lines. Finally, as a proof of concept, TGE was applied for the development of a vaccine against the respiratory syncytial virus. The fusion protein of the respiratory syncytial virus was produced by TGE and used as an antigen for the development of a recombinant vaccine. TGE enabled the rapid evaluation of the candidate vaccine in animal trials. Overall, the results of this study suggest that TGE is a reliable and reproducible method for manufacturing r-proteins of a consistent quality, provided that some particular features, such as reagents quality and process parameters, are well defined and controlled. Since TGE makes possible the rapid production of virtually any protein, even toxic, it could be used to provide GMP approved biopharmaceuticals for clinical trials in a short time, reducing development costs of biological therapeutic products.

EPFL2010

OrbShake Bioreactors for Mammalian Cell Cultures

Stéphanie Tissot

Most of the biopharmaceuticals nowadays are produced by animal cells. The worldwide demand for a specific group of biopharmaceuticals, the monoclonal antibodies (mAbs), has significantly increased this last decade. The mAbs are used as therapeutics in particular against cancer and autoimmune disorders. Most of the mAbs are produced as recombinant proteins by mammalian cells which ensure human-like post-translationnal modifications to obtain a fully functional product. However, bioprocesses with mammalian cells are usually expensive and the mAbs are needed in large amounts. As a consequence, highly flexible and cost-effective bioprocesses are required for new drug research and development as well as for the production of approved biopharmaceuticals. Orbitally shaken bioreactors (OSRs) are based on the disposable technology (single-use bioreactors) and are therefore very flexible. This work aims to improve our understanding of these bioreactors by studying their engineering principles such as mixing, the gas transfer and the hydrodynamics in the frame of mammalian cell culture. The mixing study showed that OSRs ensure homogeneity of the mammalian cell culture at scales up to 1500 L with mixing times below 30 s. By keeping the geometric ratios (shaking diameter, inner diameter and liquid height) and the Froude number constant, mixing and the free-surface shape of a 1500-L OSR could be mimicked in a 30-L OSR. A minimal volumetric mass transfer coefficient of oxygen (kLa) was required to operate mammalian cell culture without needing controllers to compensate the pH or the dissolved oxygen concentration (DO). This minimal kLa was cell-line dependent and typically in the range of 7-10 h-1. Cultures with the same kLa showed the same growth characteristics, recombinant protein concentrations, culture conditions (pH and DO), and metabolite profiles. A probe-independent bioprocess was run in a 200-L OSR at the same kLa as 1- and 5-L OSRs. Similar cell growth, recombinant protein concentration, pH and DO profiles were observed in the three OSRs. The shear stress level calculated by computational fluid dynamics (CFD) in a 1-L OSR at 110 rpm was one to two orders of magnitude lower than the one reported to damage mammalian cells. The CFD simulations showed that the maximal shear stress in OSRs was located at the tip of the wave, while most of the zones in the liquid had shear stress values below or equal to 0.075 Pa. Cell damage in a 1-L OSR was recorded at agitation rates ≥ 170 rpm which corresponded to maximal shear stress values of 0.2 Pa. Probe-independent bioprocesses in OSRs were compared side-by-side with fully controlled bioprocesses in stirred-tank bioreactors (STRs). The bioprocesses in OSRs were as efficient as those in STRs. Furthermore, a very low variability (≤ 10% for cell growth, recombinant protein concentration, pH and DO profiles) was observed for probe-independent bioprocesses in 5-L OSRs. Together, the results presented in this thesis demonstrate that probe-independent bioprocesses are scalable in OSRs from 50 mL to 100 L.

EPFL2011

The Engineering of a Scalable Disposable Orbitally Shaken Bioreactor System for Animal Cell Cultivation and Therapeutic Protein Production

Dominique Terry Monteil Jr

The aim of the pharmaceutical industry is to manufacture complex therapeutic proteins or biologics to treat human diseases. The manufacture of biologics requires a significant amount of financial and infrastructure resources. For this reason, efforts are being made in the industry to develop more effective, flexible and cost-efficient bioprocesses. This thesis focused on the development and characterization of a new disposable bioreactor for the scale-up of animal cell cultures for the production of therapeutic proteins. These disposable orbitally shaken bioreactors (OSRs) are beginning to be used in the biopharmaceutical industry and are a potential alternative to stirred-tank bioreactors (STRs) for small- and production-scale bioprocess. With cell culture volumes of mL to 2000 L available, a scale-up platform using only OSRs is feasible, but has yet to been tested. However, little is understood about OSRs above the bench-scale. Therefore, we investigated which small-scale OSR would be optimal for mammalian cell culture and then investigated a potential OSR scale-up bioprocesses that included newly developed pilot- and production-scale OSRs with working volumes of 200 L and 2500 L, respectively. When compared to the Erlenmeyer shake flask and cylindrical bottle, the TubeSpin bioreactor 600 exhibited a higher oxygen mass transfer coefficient and more efficient carbon dioxide stripping, while mammalian cell growth and recombinant protein production profiles were similar. Next, a study investigating protein quality and the effect of pH modulation in OSRs and STRs was performed in 3-L STRs and 5-L cylindrical bottles. Based on specific power consumption and mixing measurements, the OSR was found to be a more energy efficient system compared to the STR. Similar viable cell growth and recombinant protein production profiles in pH-controlled and non-controlled cultures were observed in both OSRs and STRs. However, a higher galactosylation index was observed for the recombinant protein produced in OSRs relative to that recovered from the STRs. To investigate the potential for the large-scale manufacture of recombinant proteins with OSRs, a scale-up bioprocess platform was developed for cell cultivation at scales from 10 mL to 2000 L. Similar engineering principles, mammalian cell growth, and recombinant protein production profiles were observed at all OSR scales. However, minor cell damage was found in the production-scale OSR. In order to further optimize the pilot- and production-scale OSRs, scale-down models were constructed. Wave patterns in the pilot- and production-scale OSRs were reproduced with the scale-down models. In addition, the scale-down models displayed similar engineering parameters as the pilot- and production-scale OSRs and the trends in cell growth and protein production were similar, validating the scale-down models. Subsequently, different bioprocesses were performed in small-scale OSRs in order to validate the technology for other applications. Microcarrier-based cell cultures and suspension-adapted insect cell cultures were performed using the OSR platform. In both cases, excellent cell growth was observed, demonstrating the flexibility of the OSR cell culture platform. In conclusion, the feasibility to perform large-scale bioprocesses with OSRs for the production of biologics at scales up to 2000 L has been demonstrated with animal cells in suspension and on microcarriers.

EPFL2014