Disposable bioreactor for cell culture using wave-induced agitation

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

WAVETM生物反应器灌注培养生产种子细胞的开发和应用

近年来,国内中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)生产罐的培养规模已达上千升,国外已达上万升,最终的生产罐前需要多级摇瓶、种子罐进行种子细胞扩增,扩增效率较低,严重影响了抗体、融合蛋白等生物制品的生产效率。文中利用WAVETM波浪式生物反应器,通过灌注培养的方法,成功地实现了种子细胞的高效扩增。WAVETM波浪式生物反应器灌注培养方法制备种子细胞,CHO细胞密度高达2.28×107cells/mL时仍处于指数生长期且细胞活力大于95%,以此细胞作为种子细胞,4×105cells/mL接种于另一个WAVETM生物反应器进行流加培养,最大活细胞密度仍可达1.73×107cells/mL。通过此种扩增方式,1台WAVETM20/50即可为1 000 L或2 000 L的生产罐提供种子细胞,种子细胞的扩增倍数(Split ratio)可以达到1∶50～1∶100倍,与传统不锈钢罐种子细胞扩增倍数1∶2～1∶10相比,可以显著减少2～3级种子罐,种子细胞的扩增时间减少7～9 d,极大地提高生产效率。     

A versatile modular bioreactor platform for Tissue Engineering

Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue‐specific, non‐disposable bioreactor systems. To ensure a high level of standardization, a suitable cost‐effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles. Characterization revealed improved performance of the modules in comparison to traditional cell culture equipment such as incubators, or peristaltic pumps. By combining the modules, a broad range of culture conditions can be achieved. The novel bioreactor platform allows using disposable components and facilitates tissue culture in closed fluidic systems. By sustaining native carotid arteries, engineering a blood vessel, and generating intestinal tissue models according to a previously published protocol the feasibility and performance of the bioreactor platform was demonstrated.     

Assessment of mass transfer and mixing in rigid lab‐scale disposable bioreactors at low power input levels

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied. These settings are generally used for cultivation of mammalian cells growing adherent to microcarriers. The rigid disposable vessels showed similar engineering characteristics compared to a traditional glass bioreactor. On the basis of the presented results appropriate settings for adherent cell culture, normally operated at a maximum power input level of 5 W m^?3, can be selected. Depending on the disposable bioreactor used, a stirrer speed ranging from 38 to 147 rpm will result in such a power input of 5 W m^?3. This power input will mix the fluid to a degree of 95% in 22 ± 1 s and produce a volumetric mass transfer coefficient of 0.46 ± 0.07 h^?1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1269–1276, 2014     

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality.     

Enhanced Production of carboxymethylcellulase by a marine bacterium,Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2‐L bioreactor at agitation speeds at interval 600‐1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale‐up of bioreactors mass transfer parameters were estimated and lowest values of K_La obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ‐polyglutamic acid (PGA) showed higher viability than control group.     

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Short Communication: Effect of agitation rate on the growth and production of xylanase free of cellulase by Thermomyces lanuginosus MH4 in bioreactor

Cellulase-free xylanase production by T. lanuginosus MH4 was investigated in a 3-litre stirred tank bioreactor under different agitation rates and an aeration rate of 1v/v/m. The cultivation time in the bioreactor was reduced significantly over that in shake culture conditions. A xylanase productivity of 0.1 mkat1–1h–1 was achieved on xylan in the bioreactor. This was nearly double to that obtained in shake culture. The agitation rates influenced both growth and enzyme secretion in the bioreactor. The highest level of biomass concentration and activities of both xylanase and -xylosidase were obtained at 150 revmin–1     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

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.     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014     

A simple eccentric stirred tank mini‐bioreactor: Mixing characterization and mammalian cell culture experiments

In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5–100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple—easy to assemble, easy to use, easy to clean—cell culture mini‐bioreactors for lab‐scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini‐bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini‐bioreactor were comparable to those observed for 6‐well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini‐bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini‐bioreactor. Biotechnol. Bioeng. 2013; 110: 1106–1118. © 2012 Wiley Periodicals, Inc.     

A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells?

It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. (c) 1996 John Wiley & Sons, Inc.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop