Equalizing growth in high-throughput small scale cultivations via precultures operated in fed-batch mode

This manuscript deals with the detailed design of a small digestion prototype intended as a commercial unit fully operational to cover the demand for decentralized treatment of wastes. These plants are highly affected by the complex nature of wastes giving rise to different operating problems that should be considered in detail. This paper describes the design and start-up strategy of a small-scale digestion plant with a volume of 8 m3 designed to operate with a hydrolysis pretreatment unit. The plant was designed to treat fruit and vegetable wastes as substrates derived from a local processing food factory. The performance of the plant during fed-batch operation was reported. The strategy of inoculating the reactor only to a third of its original volume and subsequently increasing the volume of the reactor by using the fed-batch mode was inadequate. The acid pH of the feeding substrate resulted in the application of a low organic loading rate with a volumetric variation of just 19.7 L/d. The performance of the plant was evaluated at non-steady state conditions and resulted in excessive destruction of volatile solids due to the low nitrogen content of the feeding substrate. The prototype reported a specific methane production of 232 L/kg volatile solids despite the low feeding rate supplemented.

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High-throughput microbioreactor provides a capable tool for early stage bioprocess development

Scientific Reports ◽

10.1038/s41598-021-81633-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mathias Fink ◽

Monika Cserjan-Puschmann ◽

Daniela Reinisch ◽

Gerald Striedner

Keyword(s):

High Throughput ◽

Process Development ◽

Early Stage ◽

Explanatory Power ◽

Batch Mode ◽

E Coli ◽

Speed Up ◽

Bioreactor System ◽

Cell Densities ◽

Microbial Systems

AbstractTremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.

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

Microbial Cell Factories ◽

10.1186/s12934-021-01541-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Tobias Habicher ◽

Tobias Klein ◽

Jacqueline Becker ◽

Andreas Daub ◽

Jochen Büchs

Keyword(s):

Controlled Release ◽

Bacillus Licheniformis ◽

Protease Activity ◽

Microtiter Plate ◽

Fed Batch ◽

Batch Mode ◽

Screening Process ◽

Microtiter Plates ◽

Batch Fermenter ◽

Production Conditions

Abstract 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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Fully Automated Cultivation of Adipose-Derived Stem Cells in the StemCellDiscovery—A Robotic Laboratory for Small-Scale, High-Throughput Cell Production Including Deep Learning-Based Confluence Estimation

Processes ◽

10.3390/pr9040575 ◽

2021 ◽

Vol 9 (4) ◽

pp. 575

Author(s):

Jelena Ochs ◽

Ferdinand Biermann ◽

Tobias Piotrowski ◽

Frederik Erkens ◽

Bastian Nießing ◽

...

Keyword(s):

Stem Cells ◽

Deep Learning ◽

High Throughput ◽

High Speed ◽

New Technologies ◽

Human Mesenchymal Stem Cells ◽

Simulation Software ◽

System Capacity ◽

Small Scale ◽

Production Environment

Laboratory automation is a key driver in biotechnology and an enabler for powerful new technologies and applications. In particular, in the field of personalized therapies, automation in research and production is a prerequisite for achieving cost efficiency and broad availability of tailored treatments. For this reason, we present the StemCellDiscovery, a fully automated robotic laboratory for the cultivation of human mesenchymal stem cells (hMSCs) in small scale and in parallel. While the system can handle different kinds of adherent cells, here, we focus on the cultivation of adipose-derived hMSCs. The StemCellDiscovery provides an in-line visual quality control for automated confluence estimation, which is realized by combining high-speed microscopy with deep learning-based image processing. We demonstrate the feasibility of the algorithm to detect hMSCs in culture at different densities and calculate confluences based on the resulting image. Furthermore, we show that the StemCellDiscovery is capable of expanding adipose-derived hMSCs in a fully automated manner using the confluence estimation algorithm. In order to estimate the system capacity under high-throughput conditions, we modeled the production environment in a simulation software. The simulations of the production process indicate that the robotic laboratory is capable of handling more than 95 cell culture plates per day.

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Optimized Large Scale Synthesis of Phosphorothioate Oligonucleotides on PS-PEG Supports

Zeitschrift für Naturforschung B ◽

10.1515/znb-1997-0122 ◽

1997 ◽

Vol 52 (1) ◽

pp. 110-116

Author(s):

Michael Gerster ◽

Martin Maier ◽

Nils Clausen ◽

Jens Schewitz ◽

Ernst Bayer

Keyword(s):

Gel Electrophoresis ◽

Continuous Flow ◽

Large Scale ◽

Electrospray Mass Spectrometry ◽

Small Scale ◽

Batch Mode ◽

Crucial Step ◽

Capillary Gel Electrophoresis ◽

Phosphoramidite Chemistry ◽

Large Scale Synthesis

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG

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Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/24726303211047839 ◽

2021 ◽

pp. 247263032110478

Author(s):

Nicolás M. Morato ◽

MyPhuong T. Le ◽

Dylan T. Holden ◽

R. Graham Cooks

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Desorption Electrospray Ionization ◽

Ambient Ionization ◽

Machine Learning Algorithms ◽

Small Scale ◽

Label Free ◽

Minimal Sample ◽

Secondary Droplets ◽

Automated Platform

The Purdue Make It system is a unique automated platform capable of small-scale in situ synthesis, screening small-molecule reactions, and performing direct label-free bioassays. The platform is based on desorption electrospray ionization (DESI), an ambient ionization method that allows for minimal sample workup and is capable of accelerating reactions in secondary droplets, thus conferring unique advantages compared with other high-throughput screening technologies. By combining DESI with liquid handling robotics, the system achieves throughputs of more than 1 sample/s, handling up to 6144 samples in a single run. As little as 100 fmol/spot of analyte is required to perform both initial analysis by mass spectrometry (MS) and further MSn structural characterization. The data obtained are processed using custom software so that results are easily visualized as interactive heatmaps of reaction plates based on the peak intensities of m/ z values of interest. In this paper, we review the system’s capabilities as described in previous publications and demonstrate its utilization in two new high-throughput campaigns: (1) the screening of 188 unique combinatorial reactions (24 reaction types, 188 unique reaction mixtures) to determine reactivity trends and (2) label-free studies of the nicotinamide N-methyltransferase enzyme directly from the bioassay buffer. The system’s versatility holds promise for several future directions, including the collection of secondary droplets containing the products from successful reaction screening measurements, the development of machine learning algorithms using data collected from compound library screening, and the adaption of a variety of relevant bioassays to high-throughput MS.

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The Scalability of Wet Ball Milling for The Production of Nanosuspensions

Pharmaceutical Nanotechnology ◽

10.2174/2211738507666190401142530 ◽

2019 ◽

Vol 7 (2) ◽

pp. 147-161 ◽

Cited By ~ 2

Author(s):

Maria L.A.D. Lestari ◽

Rainer H. Müller ◽

Jan P. Möschwitzer

Keyword(s):

Ball Milling ◽

Large Scale ◽

Milling Time ◽

High Energy ◽

Small Scale ◽

Particle Sizes ◽

Scale Production ◽

Batch Mode ◽

Pharmaceutical Ingredients ◽

Large Scale Production

Background: Miniaturization of nanosuspensions preparation is a necessity in order to enable proper formulation screening before nanosizing can be performed on a large scale. Ideally, the information generated at small scale is predictive for large scale production. Objective: This study was aimed to investigate the scalability when producing nanosuspensions starting from a 10 g scale of nanosuspension using low energy wet ball milling up to production scales of 120 g nanosuspension and 2 kg nanosuspension by using a standard high energy wet ball milling operated in batch mode or recirculation mode, respectively. Methods: Two different active pharmaceutical ingredients, i.e. curcumin and hesperetin, have been used in this study. The investigated factors include the milling time, milling speed, and the type of mill. Results: Comparable particle sizes of about 151 nm to 190 nm were obtained for both active pharmaceutical ingredients at the same milling time and milling speed when the drugs were processed at 10 g using low energy wet ball milling or 120 g using high energy wet ball milling in batch mode, respectively. However, an adjustment of the milling speed was needed for the 2 kg scale produced using high energy wet ball milling in recirculation mode to obtain particle sizes comparable to the small scale process. Conclusion: These results confirm in general, the scalability of wet ball milling as well as the suitability of small scale processing in order to correctly identify the most suitable formulations for large scale production using high energy milling.

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