Cultivation of Cells and Microorganisms in Wave‐Mixed Disposable Bag Bioreactors at Different Scales

Junne, Stefan; Solymosi, Thomas; Oosterhuis, Nico; Neubauer, Peter

doi:10.1002/cite.201200149

Cited by 35 publications

(27 citation statements)

References 46 publications

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“…This approach minimizes Fab leakage and maintains higher overall yield than cultivation with the nutrient supplementation under high aeration conditions. Since maximum Fab expression is achieved at relatively low aeration rates, Fab production at larger scale could be well accomplished in vessels such as disposable wave-mixed bioreactors [41] that have lower aeration rates compared to stirred bioreactors. The enzyme-based fed-batch system should be well suited to larger scale Fab production as it has been demonstrated well scalable up to pilot plant scale [42] and applicable to disposable bag bioreactors [43].…”

Section: Discussionmentioning

confidence: 99%

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

Ukkonen

Veijola

Vasala³

et al. 2013

Microb Cell Fact

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BackgroundFab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions.ResultsSmall-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume.ConclusionsYield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.

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Section: Discussionmentioning

confidence: 99%

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

Ukkonen

Veijola

Vasala³

et al. 2013

Microb Cell Fact

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“…Junne et al demonstrated the application of the CELL-tainer for the cultivation of Escherichia coli [11]. In these cultivations, a glucose-limited fed-batch process was successfully employed to prevent oxygen limitation.…”

Section: Cultivation Of Microbial Cells With High Oxygen Demand In Simentioning

confidence: 99%

“…Several promising approaches, such as cultivation in the CELL-tainer system, have been scaled up to near 150 L [11]. Cultivations with heterotrophic marine microorganisms were successful on the 15 L scale [105], therefore cultivations on this larger scale are envisaged.…”

Section: Summary Conclusion and Outlookmentioning

confidence: 99%

Cultivation of Marine Microorganisms in Single-Use Systems

Hillig

Pilarek

Junne

et al. 2013

Advances in Biochemical Engineering/Biotechnology

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: Marine cultures are an important source of novel substances and enzymes. As efforts to isolate strains from (deep) sea environments increase, the demand for methodology platforms to cultivate these organisms is also rising. Due to the high salt concentration and the shear sensitivity exhibited by some heterotrophic microalgae, single-use systems originally designed for the cultivation of mammalian cell lines can be a valuable alternative. Using the cultivation of the heterotrophic marine microalgae Crypthecodinium cohnii as an example, this chapter makes suggestions for experimental design, for improving process development by integrating parallel experiments, and for scaling-up and scaling-down methodologies. It describes how to identify suitable single-use systems and how to integrate a two-layer system with perfluordecalin to improve the gas transfer in deep-well plates. The process is also scaled up in several single-use systems. We also describe challenges in the process development to achieve sufficient oxygen transfer, monitoring, and control, and we discuss limitations such as corrosion, long-term stability, and leachables in single-use systems. Finally, we demonstrate a method for cheap, fast, and consistent process development for marine microorganisms.

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“…Even new formats that propose improved mixing and mass transfer, like e.g. the BayShake R (<90 s, worst case conditions 4 W m −3 and no submerse gassing [26] or Biostat Cultibag R (65 to 800 s at 200 L cultivation volume), due to the geometric shape and mode of operation [34] achieve very similar or slower mixing times.…”

Section: Mixing Times In Different Bioreactor Systemsmentioning

confidence: 99%

Harmonization and characterization of different single‐use bioreactors adopting a new sparger design

Minow

Seidemann

Tschoepe

et al. 2014

Engineering in Life Sciences

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In mammalian cell culture, single-use bioreactors are widely used. Different hardware designs are available, ranging from stirred tank reactors to wave mixed and cubical shaken systems. Unlike in stainless steel systems, where standards exist, in singleuse bioreactors aeration devices are often predefined by the supplier. While ring sparger systems are the gold standard in stainless steel bioreactors, not all singleuse bioreactors are available with ring spargers. In this study, a comprehensive characterization of two stirred tank single-use bioreactor systems (XDR TM from Xcellerex and S.U.B. from Thermo Scientific Hyclone) was performed under GMP conditions with 200/250 L and 1000 L bioreactors. Engineering facts like mass transfer rates for oxygen k L a o2 and carbon dioxide k L a CO2 as well as mixing number were evaluated. To achieve improved similarity in key engineering parameters and in consequence cell culture performance, the submerse aeration device of the S.U.B.(to date only open tube and frit) was remodeled resulting in a drilled hole sparger design. Results of the characterization showed that k L a o2 in the S.U.B. was enhanced from 8.5 h −1 to 11.5 h −1 at the maximum, and the k L a CO2 was very similar between both bioreactor types. Knowledge of the characterization data as well as improved oxygen transfer rate in the S.U.B. allows for an interchangeable usage of the two different single-use bioreactors.

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Cultivation of Cells and Microorganisms in Wave‐Mixed Disposable Bag Bioreactors at Different Scales

Cited by 35 publications

References 46 publications

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

Cultivation of Marine Microorganisms in Single-Use Systems

Harmonization and characterization of different single‐use bioreactors adopting a new sparger design

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