Optimization of antibody fragment production in Bacillus megaterium: The role of metal ions on protein secretion

David, Florian; Westphal, Ralf; Bunk, Boyke; Jahn, Dieter; Franco‐Lara, Ezequiel

doi:10.1016/j.jbiotec.2010.07.023

Cited by 21 publications

(9 citation statements)

References 44 publications

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“…In most applications of parallel small scale cultivations the MTP for cultivation is filled manually instead of doing this automatically [35,36]. Even for higher throughput this is typically not a limiting step, since it can be achieved quickly using multi-channel pipettes or semi-automated lab dispensers if the same medium is used for all cultivations.…”

Section: Resultsmentioning

confidence: 99%

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

et al. 2012

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BackgroundHigh-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest.ResultsTo increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale.ConclusionsThe optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation.

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Section: Resultsmentioning

confidence: 99%

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

et al. 2012

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“…In all experiments the B. megaterium strain YYBm1 [16] carrying the plasmid pEJBmD1.3 scFv was used [19]. For the preculture and main culture of B. megaterium a minimal medium was applied [23]. It contains fructose as a sole carbon source supplemented with 3.52 g L -1 KH 2 PO 4 , 5.297 g L -1 Na 2 HPO 4 , 3 g L -1 MgSO 4 × 7 H 2 O, 25 g L -1 (NH 4 ) 2 SO 4 , 0.312 g L -1 MnCl 2 × 4 H 2 O, 0.0954 g L -1 CaCl 2 × 2 H 2 O, 5.5 mg L -1 FeSO 4 × 7 H 2 O, 21.6 mg L -1 (NH 4 ) 6 Mo 7 O 24 × 4 H 2 O, 6.2 mg L -1 CoCl 2 , 16 μg L -1 CuSO 4 × 5H 2 O, 155 μg L -1 H 3 BO 3 , 15 μg L -1 ZnSO 4 × 7H 2 O and 10 mg L -1 tetracycline.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a minimal medium of high production and secretion capacities could be developed based on a genetic algorithm approach varying metal ion concentrations [23]. Based on this minimal medium, batch cultivations were carried out and single cell analysis under controlled bioprocess conditions was performed to study the D1.3 scFv ABF production with B. megaterium at different stages of the cultivation.…”

Section: Introductionmentioning

confidence: 99%

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

et al. 2011

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BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

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“…The Gram-positive bacteria Bacillus brevis (76, 77), Bacillus subtilis (78, 79), and Bacillus megaterium (80–85) have already been successfully used for the production of different antibody fragments. In addition, B. megaterium does not produce alkaline proteases and provides high stability of plasmid vectors during growth allowing stable transgene expression during long term cultivation in bioreactors (86).…”

Section: Antibody Production In Prokaryotic Hostsmentioning

confidence: 99%

Expression of Recombinant Antibodies

Frenzel¹,

Hust²,

Schirrmann³

2013

Front. Immunol.

281

220

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Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

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Optimization of antibody fragment production in Bacillus megaterium: The role of metal ions on protein secretion

Cited by 21 publications

References 44 publications

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

Expression of Recombinant Antibodies

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