Glucose‐containing polymer rings enable fed‐batch operation in microtiter plates with parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH

Habicher, Tobias; Czotscher, Vroni; Klein, Tobias; Daub, Andreas; Keil, Timm; Büchs, Jochen

doi:10.1002/bit.27077

Cited by 9 publications

(9 citation statements)

References 50 publications

(67 reference statements)

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“…These culture volumes correspond to glucose release rates in the range from 0.75 g/L/h for 1.1 mL to 1.38 g/L/h for 0.6 mL filling volume, respectively (Additional file 1 ). For culture volumes higher than 0.8 mL, the OTR curves are typical for cultivations with fed-batch MTPs [ 14 , 36 , 37 ]. Initially, a batch phase of 3 h with exponential growth is observed due to accumulated glucose.…”

Section: Resultsmentioning

confidence: 99%

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: 99%

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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“…The ambr 250 was successfully demonstrated as a representative scale-down model for two mAbs commercial processes at scales of >10,000 l (Manahan et al, 2019). A polymer-based controlled-release fed-batch microtiter plate (48-well-plate) with on-line monitoring capabilities was also studied with E. coli for screenings and initial process development (Habicher et al, 2019a). 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.…”

Section: High-throughput Cultivation Systemsmentioning

confidence: 99%

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

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

397

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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“…Since Aspergillus as well as most other fungal species can metabolize various sugar polymers, the FeedER technology 38 or other enzymatic slow-release systems 39,40 are not applicable in the present case. Only recently, a further strategy enabling MBR fed-batch cultivation has been published: Through integration of polymer rings, diffusion-based fed-batch cultivation could be performed in MTPs without losing the possibility of conducting online measurements through the plate bottom 41 as is the case for standard MTP systems for diffusion-based slow-release. 42 However, for the cultivation of A. niger it seems advantageous to utilize FlowerPlates to increase shear forces toward microfilamentous morphology and to provide the mass transfer needed to avoid an oxygen limitation.…”

Section: Development Of Fed-batch Processes For Microscale Cultivatio...mentioning

confidence: 99%

Microbioreactor‐assisted cultivation workflows for time‐efficient phenotyping of protein producing Aspergillus niger in batch and fed‐batch mode

Jansen

Morschett

Hasenklever

et al. 2021

Biotechnol Progress

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In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products.However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for timeefficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via noninvasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fedbatch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.

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Glucose‐containing polymer rings enable fed‐batch operation in microtiter plates with parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH

Cited by 9 publications

References 50 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

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

Microbioreactor‐assisted cultivation workflows for time‐efficient phenotyping of protein producing Aspergillus niger in batch and fed‐batch mode

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