Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Keil, Timm; Landenberger, Markus; Dittrich, Barbara; Selzer, Sebastian; Büchs, Jochen

doi:10.1002/biot.201800727

Cited by 14 publications

(20 citation statements)

References 42 publications

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“…In all cases, an equal amount of glucose was released into each well at every point of time and the glucose release rate is equal as well. Thus, similar biomass concentrations and physiological state of the cells are expected [ 42 ].…”

Section: Resultsmentioning

confidence: 98%

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Keil

Dittrich

Lattermann³

et al. 2020

J Biol Eng

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Background Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24–0.90 mg/h per well. Conclusions The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

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Section: Resultsmentioning

confidence: 98%

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Keil

Dittrich

Lattermann³

et al. 2020

J Biol Eng

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“…With unequal growth, individual clones are in uneven growth phases at the time of harvest, but are also exposed to the stationary phase for different lengths of time. This results in different clone rankings [ 29 ]. Variations in growth in the main culture are mainly caused by differences in the initial biomass concentration and in the duration of the lag-phase.…”

Section: Resultsmentioning

confidence: 99%

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

et al. 2021

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Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

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“…In a study by Hou et al (2019), it was reported that the phosphorylation and hydroxylation level of an Fc-fusion protein could be reduced by nutrient optimization in a CHO fed-batch process. The application of fed-batch MTPs for HTP screening of E. coli clones (32 strains) was also established (Keil et al, 2019). Batch and exponential-fed-batch cultures were designed to evaluate the effect of the specific growth rate (µ) and resulted in recombinant glycoprotein AcrA glycosylation and a maximum specific synthesis rate at µ max (Caillava et al, 2019).…”

Section: Transgenic Animalsmentioning

confidence: 99%

“…Cultivations in standard 96-well-microtiter plates represent the optimal system for miniaturization (Duetz, 2007;Baumann and Hubbuch, 2017). MTPs were successfully used for recombinant protein process development (Long et al, 2014;Chung et al, 2018;Fink et al, 2019;Keil et al, 2019).…”

Section: High-throughput Cultivation Systemsmentioning

confidence: 99%

“…In a study by Fink et al (2019), 32 production clones were characterized in carbon-limited microbioreactor cultivations (BioLector), with production yields of 0-7.4 mg Fab per gram of cell dry mass. The use of polymer-based controlled-release fed-batch microtiter plates during preculture was theoretically studied and tested with an E. coli clone bank containing 32 strains (Keil et al, 2019). The suitability of an automated smallscale bioreactor (ambr 15 with 48 wells) as a small-scale model was confirmed using a perfusion process (Janoschek et al, 2019).…”

Section: High-throughput Cultivation Systemsmentioning

confidence: 99%

See 1 more Smart Citation

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

361

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Cited by 14 publications

References 42 publications

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

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