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Hemmerich J.,m2p-labs | Adelantado N.,Autonomous University of Barcelona | Barrigon J.M.,Autonomous University of Barcelona | Ponte X.,Autonomous University of Barcelona | And 5 more authors.
Microbial Cell Factories | Year: 2014

Background: In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption.Results: An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved.Conclusion: The RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies. © 2014 Hemmerich et al.; licensee BioMed Central Ltd.


Funke M.,RWTH Aachen | Buchenauer A.,RWTH Aachen | Schnakenberg U.,RWTH Aachen | Mokwa W.,RWTH Aachen | And 5 more authors.
Biotechnology and Bioengineering | Year: 2010

In industrial-scale biotechnological processes, the active control of the pH-value combined with the controlled feeding of substrate solutions (fed-batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small-scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale-up and scale-down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small-scale batches are typically performed highly parallel and in high throughput, large-scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber-optic online-monitoring device for microtiter plates (MTPs)-the BioLector technology-together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed-batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user-friendly and can easily be transferred to a disposable single-use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH-controlled and fed-batch conditions in shaken MTPs. © 2010 Wiley Periodicals, Inc.


Funke M.,RWTH Aachen | Buchenauer A.,RWTH Aachen | Mokwa W.,RWTH Aachen | Kluge S.,RWTH Aachen | And 4 more authors.
Microbial Cell Factories | Year: 2010

The efficiency of biotechnological production processes depends on selecting the best performing microbial strain and the optimal cultivation conditions. Thus, many experiments have to be conducted, which conflicts with the demand to speed up drug development processes. Consequently, there is a great need for high-throughput devices that allow rapid and reliable bioprocess development. This need is addressed, for example, by the fiber-optic online-monitoring system BioLector which utilizes the wells of shaken microtiter plates (MTPs) as small-scale fermenters. To further improve the application of MTPs as microbioreactors, in this paper, the BioLector technology is combined with microfluidic bioprocess control in MTPs. To realize a user-friendly system for routine laboratory work, disposable microfluidic MTPs are utilized which are actuated by a user-friendly pneumatic hardware.Results: This novel microfermentation system was tested in pH-controlled batch as well as in fed-batch fermentations of Escherichia coli. The pH-value in the culture broth could be kept in a narrow dead band of 0.03 around the pH-setpoint, by pneumatically dosing ammonia solution and phosphoric acid to each culture well. Furthermore, fed-batch cultivations with linear and exponential feeding of 500 g/L glucose solution were conducted. Finally, the scale-up potential of the microscale fermentations was evaluated by comparing the obtained results to that of fully controlled fermentations in a 2 L laboratory-scale fermenter (working volume of 1 L). The scale-up was realized by keeping the volumetric mass transfer coefficient kLa constant at a value of 460 1/h. The same growth behavior of the E. coli cultures could be observed on both scales.Conclusion: In microfluidic MTPs, pH-controlled batch as well as fed-batch fermentations were successfully performed. The liquid dosing as well as the biomass growth kinetics of the process-controlled fermentations agreed well both in the microscale and laboratory scale. In conclusion, a user-friendly and disposable microfluidic system could be established which allows scaleable, fully controlled and fully monitored fermentations in working volumes below 1 milliliter. © 2010 Funke et al; licensee BioMed Central Ltd.


Huber R.,RWTH Aachen | Palmen T.G.,RWTH Aachen | Ryk N.,RWTH Aachen | Hillmer A.-K.,RWTH Aachen | And 3 more authors.
BMC Biotechnology | Year: 2010

Background: High-throughput cultivations in microtiter plates are the method of choice to express proteins from recombinant clone libraries. Such processes typically include several steps, whereby some of them are linked by replication steps: transformation, plating, colony picking, preculture, main culture and induction. In this study, the effects of conventional replication methods and replication tools (8-channel pipette, 96-pin replicators: steel replicator with fixed or spring-loaded pins, plastic replicator with fixed pins) on growth kinetics of Escherichia coli SCS1 pQE-30 pSE111 were observed. Growth was monitored with the BioLector, an on-line monitoring technique for microtiter plates. Furthermore, the influence of these effects on product formation of Escherichia coli pRhotHi-2-EcFbFP was investigated. Finally, a high-throughput cultivation process was simulated with Corynebacterium glutamicum pEKEx2-phoD-GFP, beginning at the colony picking step.Results: Applying different replication tools and methods for one single strain resulted in high time differences of growth of the slowest and fastest growing culture. The shortest time difference (0.3 h) was evaluated for the 96 cultures that were transferred with an 8-channel pipette from a thawed and mixed cryoculture and the longest time difference (6.9 h) for cultures that were transferred with a steel replicator with fixed pins from a frozen cryoculture. The on-line monitoring of a simulated high-throughput cultivation process revealed strong variances in growth kinetics and a twofold difference in product formation. Another experiment showed that varying growth kinetics, caused by varying initial biomass concentrations (OD600of 0.0125 to 0.2) led to strongly varying product formation upon induction at a defined point of time.Conclusions: To improve the reproducibility of high-throughput cultivation processes and the comparability between different applied cultures, it is strongly recommended to use automated or manual liquid handling stations or, alternatively, multi-channel pipettes. Because of their higher transfer volume and hence precision in comparison to pin replicators, they reduce the variance of initial biomass concentrations. With respect to the results obtained, other methods to increase the comparability between parallel cultivations by compensating differences in biomass concentrations are required, such as using autoinduction media, fed-batch operation of precultures or on-line monitoring in microtiter plates combined with automated liquid handling. © 2010 Huber et al; licensee BioMed Central Ltd.


Kottmeier K.,RWTH Aachen | Kottmeier K.,TU Dresden | Muller C.,RWTH Aachen | Muller C.,m2p-labs | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2010

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source. © 2009 Springer-Verlag.


Patent
m2p-labs | Date: 2014-10-07

A device and method for the individual dosing or drainage of small quantities of liquids or gases in microreactors and microreactor arrays, haying a membrane and an outlet to a reaction chamber or a reactor arranged therebelow, which is surrounded by an area which forms a fluid line with the membrane, wherein the area or the membrane has an annular elevation which can be arranged around the outlet.


Wenk P.,m2p-labs | Hemmerich J.,m2p-labs | Muller C.,m2p-labs | Kensy F.,m2p-labs
Chemie-Ingenieur-Technik | Year: 2012

The efficiency of fermentation processes is a key factor for the success of modern biotechnology. New designed flower-shaped microplates (Flowerplate) with integrated online sensors offer new possibilities to gain a deeper process understanding in the early stages of bioprocess development. Additionally, automation of the mass transfer-optimized Flowerplates in the RoboLector provides the potential of a systematic, high-throughput approach to study bioprocesses and promises to reduce time to market. This review gives an overview about the current state of the art in shaken microbioreactors with online monitoring and their automation approach. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Trademark
m2p-labs | Date: 2012-09-15

Motors and engines except for land vehicles; machine couplings and transmission components except for land vehicles; incubators for eggs; automatic vending machines. Electric laboratory apparatus and instruments, namely, analysis instruments for cell analysis, laboratory robots, namely, pipetting robots; micro bioreactors for laboratory fermentation; automated laboratory equipment for automated machines for examining cells and biochemical and chemical bioprocess reactions; data processing equipment, namely, computers; software for operating automated laboratory equipment; research laboratory analyzers for measuring, testing and analyzing cells; research lab mixing apparatus for mixing liquids; laboratory automated instruments for evaluating chemical, biological and biochemical reactions; automated laboratory experimentation platforms for media preparation, automated sampling, induction and fed-batch processing of biomolecules; measurement apparatus and instruments, scientific instruments for shaking, agitation apparatus, analysis apparatus, all for biochemical, biological and chemical reactions in the nature of laboratory equipment and supplies; laboratory incubators which incorporate agitation media, electric or electronic sensors for measuring temperatures and biochemical reactions; electronic controllers for operating laboratory machines, and computer hardware and software for application in basic research, screening and process development of biochemicals, recombinant drugs and biomolecules; machines, namely, micro bioreactors and micro fermentation units and machine tools, namely, laboratory pipetting robots. Conducting chemical and biological analysis.


News Article | November 7, 2013
Site: www.finsmes.com

The round was led by new investor Fidura Private Equity Fonds (which acquired a total stake of 27.61% in the company), with participation from High-Tech-Gründerfonds. The company intends to use the funds to further expand its technology with new developments and product improvements, particularly in the field of microscale process control (fed-batch and pH control) and automation of its BioLector®, and to increase its global sales network, especially in the USA and Asia. Co-founded in 2005 by and Frank Kensy and Carsten Müller, m2p-labs (“m2p” stands for “from microreactor to process”) is engaged in the development and sales of lab analysis systems (microbioreactors) to carry out high throughput test series in the field of cellular screening and bioprocess development. The company focuses on microreaction and automated solutions for screening and bioprocess development. Commercialized products include the BioLector® and the FlowerPlate®, which provide an intelligent micro fermentation platform to enable the biotechnology, chemical and pharmaceutical industry to increase their number and information of microbial and cell culture experiments. Customers include pharmaceutical and chemical companies, universities (ETH Zürich, RWTH Aachen, University College London and Imperial College London) and research institutions such as the Jülich Research Centre.

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