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 5 L OSR and a 3 L 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. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174–1180, 2016     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Somatic embryogenesis of pepper in bioreactors: a study of bioreactor type and oxygen-uptake rates

Somatic embryogenesis of pepper, Capsicum annuum var. Ace, was performed in an airlift bioreactor and a magnetically stirred hanging-stirrer-bar bioreactor, each with 1.81 working volume. All stages of embryogenesis, from growth of embryogenic suspension cultures to embryo maturation, were performed in the bioreactor as a series of drain-and-fill batches, keeping the cells and embryos in the bioreactor all the time. When two bioreactors were compared in terms of percentage embryogenesis and visually observed quality of mixing, under different rates of aeration and stirring, the performance of the magnetically stirred bioreactor was better. The effects of inoculum type and inoculum level on the percentage embryogenesis were also investigated. Under the optimum conditions, embryogenesis was 98%, with 57 embryos/ml. Oxygen-uptake rates of cultures in different stages of embryogenesis were different, the highest being in the embryogenic suspension culture and the lowest during embryo maturation.     

Thermostable lipolytic enzymes production in batch and continuous cultures of Thermus thermophilus HB27

Several studies in laboratory-scale bioreactors are undertaken in order to verify the beneficial effect of thermal spring water in the culture medium of Thermus thermophilus HB27. Two bioreactor configurations, stirred tank and airlift, are investigated to determine the most suitable one to develop a continuous process. Water mineral composition affects the lipolytic enzyme secretion and growth of T. thermophilus HB27 in both bioreactor configurations. Furthermore, the lipolytic activity is strongly enhanced when stirred tank bioreactor is used. Moreover, operation in a stirred tank at an agitation rate of 650 rpm leads to the highest total lipolytic activity (intra- and extracellular enzyme) around 280 U/L after 32 h. Continuous cultures operating in the optimised conditions determined in batch cultures are carried out. It is noticeable that the stirred tank bioreactor was able to operate in a continuous flow mode without operational problems. In addition, the lipolytic activity obtained is about 2-fold higher than that attained in batch cultures.     

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-β-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C₁ metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.     

Production of carrot somatic embryos in a bioreactor

Somatic carrot embryos were grown as batch cultures in a stirred 10-l bioreactor. Embryo production in the bioreactor was comparable to that obtained in shake-flasks. A production of about 50·10³ embryos/l per day was commonly achieved with an inoculum density of 0.1% volume of tissues/volume of medium. Regularly changing of the medium increased embryo viability. A filtering unit coupled to the bioreactor was developed in order to calibrate embryos. The characteristics of the population of harvested embryos are described. Correspondence to: J.-P. Ducos     

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Bioreactor studies on hairy root cultures of Catharanthus roseus: Comparison of three bioreactor types

Summary Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors. The best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing. In the stirred bioreactor or in the bioreactor with medium circulation the roots did not grow, suggesting that hairy roots of C. roseus are more sensitive to stress than root cultures of many other plant species.     

Advances in clone selection using high‐throughput bioreactors

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench‐scale is limited by the scale‐up and operational costs involved. This study describes the application of miniaturized stirred high‐throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

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.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis

Protocorm-like bodies (PLBs) formed on leaf segmentsin vitro were used as explants for bioreactor cultures. Continuous immersion cultures (air lift column and air lift-balloon bioreactor), and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections. A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. About 18,000 PLBs were harvested from 20 g of inoculum (∼1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Aeration in a bioreactor at 0.5 or 2.0 volume of air per volume of medium min⁻¹ (vvm) yielded similar levels of biomass production. PLBs grown in bioreactors were cultured on solid Murashige and Skoog, Vacin and Went, Knudson C, Lindemann and Hyponex media. Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors, rate of proliferation, and regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transient responses of continuously growing yeast cultures to dilution rate shifts: a sensitive means to analyze biology and the performance of equipment

Transient responses of continuously growing yeast cultures to step changes in dilution rate were influenced by (1) the preshift conditions of the culture, (2) the magnitude of the dilution rate increase, (3) the organism involved, and (4) the type of bioreactor used. The sensitivity of transient responses to seemingly minor differences in the design of experiments indicated that this type of investigation may be a means to analyze kinetics of microbial cultures as well as the effectiveness of bioreactors.     

Influence of growth rate on the accumulation of ergosterol in yeast-cells

Summary The influence of growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose, maltose, ethanol and acetic acid as substrates under C- and N-limitations in chemostat experiments. In carbon limited cultures an decrease in ergosterol content with rising dilution rate was observed, whereas in nitrogen limited cells an quite opposite behaviour was attained. A maximum specific rate of ergosterol synthesis of about 2 mg per h per g dry cell mass was calculated for nitrogen limited cultures.     

Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism and product formation.

A laboratory scale bioreactor was constructed from glass and polycarbonate materials whereby a track-etch membrane (3 microns pore diameter) was integrated into its two-part bottom flange. The reactor performance was evaluated for continuous hybridoma suspension cultures under various conditions of cell retention. A total retention experiment demonstrated that this type of stirred tank reactor cannot be operated at near zero growth rate conditions. Instead, at steady viable cell concentrations of congruent to 3 x 10(6) cells per ml, specific growth and death rates were estimated at 0.60 +/- 0.06 d-1. Specific substrate (glucose, glutamine, O2, amino acids) consumption, by-product (ammonia, alanine, amino acids) and product (antibody) production rates as well as various apparent molar yield coefficients were obtained and are compared to metabolic quotients and yield coefficients previously calculated from standard continuous culture experiments, i.e., without cell retention, at specific growth rates of 0.63 and 1.24 d-1. Furthermore, steady-state data on viable cell and antibody concentrations, spec. mAb productivities, and space-time yields determined before and after a step change (2.5-fold increase) in dilution rate at identical specific growth rates mu are presented.     

Bioactive metabolite production and stress-related hormones in Devil’s claw cell suspension cultures grown in bioreactors

In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems.     

Influence of growth rate on the accumulation of ergosterol in yeast-cells in a phosphate limited continuous culture

Summary The influence of the growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose and ethanol as substrates under P-limitation in chemostat experiments. In cultures with glucose as carbon source a decrease in ergosterol content with dilution rates up to 0.08 h^–1 was observed, whereas above this dilution rate an increase in ergosterol content occurred. Similar but less marked effects were attained with ethanol as carbon source. A maximum specific rate of ergosterol synthesis of about 2.4 mg per h and g dry cell mass was calculated for phosphorus limited cultures.