On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O₂ (DO) and dissolved CO₂ (dCO₂) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO₂ in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO₂ at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.     

Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers

Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.     

Noninvasive online biomass detector system for cultivation in shake flasks

A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°‐scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichia coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD₆₀₀ ≤ 30. The determination of online OD showed relative errors of about 7.5% for E. coli K12 and 12% for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

On line estimation of oxygen uptake rate for insect cells growing in a modified spinner flask

The simple design of traditional spinner flasks makes the on-line estimation of cellular metabolism impossible. An on-line estimation system has been developed and used for the monitoring of oxygen uptake rate (OUR) for insect cells growing in a modified spinner flask. Neglect of oxygen desorption from culture media is a common source of error in OUR measurements for Sf21 cells. Therefore, an algorithm was developed to compensate for the affect of such desorption process on the determination of OUR. A modified spinner flask was successfully used as a low-volume bioreactor for insect cell cultivation and the OUR measurement developed here is both convenient and reliable.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Application of process mass spectroscopy to the detection of metabolic changes in plant tissue culture

There is a clear need in the area of plant cell culture for methods of on-line estimation of culture parameters. The introduction of plant cells into culture can result in a loss of their photoautotrophic character so that they are largely heterotrophic. As a result, fermentation off-gas analysis may not be confounded by photosynthetically-related O₂ production. In this study performance of a suspension culture of Syringa vulgaris, in a pneumatically agitated bioreactor of in-house design, was investigated. The effect of light on growth, carbohydrate metabolism and the respiratory quotient (RQ), determined by process mass spectroscopy, was studied. Yield coefficients for cells grown in the light and dark were similar although the patterns of carbohydrate uptake were quite different. Maximum biomass yields were higher in this bioreactor than normally observed in shake flasks. The RQ was dynamic during the course of the fermentation, peaking during the transition from the lag phase to the growth phase. It is suggested that the RQ may prove useful as an on-line parameter for monitoring transitions in cellular metabolism during plant cell culture fermentations.Abbreviations RQ respiratory quotient - v.v.m. volume of gas fed to fermenter per unit volume per minute - Y_X/S growth yield coefficient based on total carbohydrate     

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10⁶ cells/ml, maximal cell densities of 16 × 10⁶ and 6 × 10⁶ cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5 × 10⁶ cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10⁶ cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production.     

Real-time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control

Dissolved carbon dioxide (dCO₂) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO₂ in bioprocesses. A silicone sampling probe that allows the diffusion of CO₂ through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO₂ that had diffused into the probe from the culture broth (sensor was calibrated using air as zero-point calibration). The gas inside the probe was then allowed to recirculate through gas-impermeable tubing to a CO₂ monitor. We have shown that by measuring the initial diffusion rate of CO₂ into the sampling probe we were able to determine the partial pressure of the dCO₂ in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO₂ in real-time. Using the proposed sensor, we successfully implemented a dCO₂-based control scheme, which resulted in significant improvement in process performance.     

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

Summary It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×10⁶ ml^–1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml^–1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml^–1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

WAVETM反应器和搅拌瓶中微载体球转球放大培养的比较

目的:哺乳动物细胞目前已广泛用于生物工程药物如单抗和疫苗的生产.而用于贴壁细胞规模化培养的微载体,也应时应需得以开发并应用于生物制药.贴壁细胞微载体培养在搅拌罐和WAVETM反应器中都能进行.而如要进行进一步的放大培养,球转球工艺不可或缺.为了发展球转球这一新的放大技术,以及考量WAVETM反应器这种新型大规模培养设备的应用性,大量的细胞培养和球转球实验在WAVETM反应器和搅拌瓶中进行.收集到的数据得以分析比较.方法:将Vero细胞分别接入WAVETM反应器和搅拌瓶中用微载体Cytodex 1进行培养.适当补充营养并控制温度、pH等培养条件使细胞增殖.长满微载体的细胞用清洗、消化等球转球工艺的一系列步骤而分离,并放大接种到新的培养体系.球转球工艺的有效性通过记录并统计分析细胞消化分离的回收率,以及细胞重新接种生长的存活力来评估.结果:统计学分析比较WAVETM反应器和搅拌瓶中得到的细胞分离回收率分别是67.56％和39.39％,数理统计P值小于0.0003;细胞重新接种存活率分别是95.17％和78.45％,P值等于0.0107.结论:在WAVETM反应器中进行的球转球放大工艺,其总体表现和有效性远高于在搅拌瓶中得到的结果.在WAVETM反应器中培养的Vero细胞有很好的细胞状态,作为种子链和生产用罐相比搅拌型反应罐均有很大的优越性.     

Integrating a 250 mL-spinner flask with other stirred bench-scale cell culture devices: a mass transfer perspective

The bioprocess development cycle is a complex task that requires a complete understanding of the engineering of the process (e.g., mass transfer, mixing, CO(2) removal, process monitoring, and control) and its affect on cell biology and product quality. Despite their widespread use in bioprocess development, spinner flasks generally lack engineering characterization of critical physical parameters such as k(L)a, P/V, or mixing time. In this study, mass transfer characterization of a 250-mL spinner flask using optical patch-based sensors is presented. The results quantitatively show the effect of the impeller type, liquid filling volume, and agitation speed on the volumetric mass transfer coefficient (k(L)a) in a 250-mL spinner flask, and how they can be manipulated to match mass transfer capability at large culture devices. Thus, process understanding in spinner flasks can be improved, and these devices can be seamlessly integrated in a rational scale-up strategy from cell thawing to bench-scale bioreactors (and beyond) in biomanufacturing.     

Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.     

Production of mosquito densonucleosis viruses by Aedes albopictus C6/36 cells adapted to suspension culture in serum-free protein-free media

Mosquito densonucleosis viruses (MDVs) have the potential for use as biocontrol agents. To facilitate densovirus production, the Aedes albopictus mosquito cell line C6/36 was adapted to two commercially available serum-free protein-free media (SFPFM), Sf-900 II and Drosophila-SFM. Cells adapted more slowly to growth in Sf-900 II medium, but once adapted, they grew more rapidly and appeared healthier than cells growing in Drosophila-SFM. Cells that were adapted to growth in each of these SFPFM were tested for their ability to be transfected and infected with MDVs. The Sf-900 II-adapted cell line survived transfection and showed infection rates comparable with cells growing in L15 supplemented with 10% fetal bovine serum. Cells adapted to Drosophila-SFM were less infectable and did not survive transfection. Cells adapted to each of these SFPFM were adapted to growth in spinner flasks. Cells in Sf-900 II grew substantially better in spinner flasks than cells in Drosophila-SFM media. Cells grown in Sf-900 II could be frozen and, when thawed, could support the production of densonucleosis viruses in spinner flasks.     

Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

Changes in soil CO2 and O2 concentrations when radiata pine is grown in competition with pasture or weeds and possible feedbacks with radiata pine root growth and respiration

This study examined the reciprocal effects of growing ryegrass, lotus and other weed species in competition with radiata pine on soil CO₂ and O₂ concentrations and on the growth and root respiration of the radiata pine. Soil O₂ concentrations decreased and soil CO₂ concentrations increased with increasing soil depth. Radiata pine plus competing species slightly reduced soil O₂ concentrations and markedly increased soil CO₂ concentrations (up to 40 mmol mol⁻¹) compared with radiata pine alone. The dry weights of shoots and roots, and the root respiration rates of radiata pine grown with competing vegetation were much less than those for radiata pine alone. This probably was not solely caused by competition for nutrients water or light since adequate water and nutrients were supplied to all treatments and the radiata pine overtopped the competing vegetation. When radiata pine roots were raised in NaHCO₃ solutions equivalent to a range of CO₂ concentrations, succinate dehydrogenase activity (a metabolic indicator of mitochondrial respiration) and elongation rates of roots decreased as CO₂ concentrations increased from 0 to 40 mmol mol⁻¹. This suggests that the elevated CO₂ concentrations found in the experiments in soil was the cause, at least in part, of the reduced growth of radiata pine in competition with other species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioreactor magnetic fields do not affect growth and antibody productivity of GAP-A3 hybridomas

Summary GAP-A3 hybridoma cells grown in T-flasks exposed to the rotating magnetic field of a spinner flask stirrer base show no effect of stirrer speed in the range 0–150 rpm on their growth rate or antibody productivity. Similarly mechanically and magnetically driven spinner flasks at 48 and 70 rpm show no difference in these rates that may be attributed to the magnetic field.