Online monitoring in continuously shaken microtiter plates for scalable upstream bioprocessing [Elektronische Ressource] / Frank Torsten Kensy
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Online monitoring in continuously shaken microtiter plates for scalable upstream bioprocessing [Elektronische Ressource] / Frank Torsten Kensy

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Online Monitoring in Continuously Shaken Microtiter Plates for Scalable Upstream Bioprocessing Von der Fakultät für Maschinenwesen der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Ingenieurwissenschaften genehmigte Dissertation vorgelegt von Frank Torsten Kensy aus Langenfeld, Rheinland Berichter: Universitätsprofessor Dr.-Ing. Jochen Büchs sprofessor Dr. rer.nat. Gerd Gellissen Tag der mündlichen Prüfung: 16. Juni 2010 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Abstract Online Monitoring in Continuously Shaken Microtiter Plates for Scalable Upstream Bioprocessing This thesis is focusing on the applicability of microtiter plates as platform for high-throughput experimentation in bioprocess development. Therefore, three main aspects have been studied in this thesis: 1) characterization of mass transfer in microtiter plates (MTPs), 2) development of a new online-monitoring technique for detecting the most relevant fermentation parameters, 3) proof of scalabilty from microtiter plates to stirred tank fermenters. The oxygen mass transfer into microtiter plates was characterized with the sulfite oxidation method in 24-, 48- and 96-well MTPs. On the one hand, the results pointed out that the achieved maximum oxygen transfer capacities (OTR ) of 0.039 maxand 0.

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Publié par
Publié le 01 janvier 2011
Nombre de lectures 19
Langue English
Poids de l'ouvrage 6 Mo

Extrait






Online Monitoring in Continuously Shaken Microtiter Plates
for Scalable Upstream Bioprocessing





Von der Fakultät für Maschinenwesen der
Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades
eines Doktors der Ingenieurwissenschaften genehmigte Dissertation


vorgelegt von

Frank Torsten Kensy
aus Langenfeld, Rheinland





Berichter: Universitätsprofessor Dr.-Ing. Jochen Büchs
sprofessor Dr. rer.nat. Gerd Gellissen

Tag der mündlichen Prüfung: 16. Juni 2010

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Abstract
Online Monitoring in Continuously Shaken Microtiter Plates
for Scalable Upstream Bioprocessing
This thesis is focusing on the applicability of microtiter plates as platform for high-throughput
experimentation in bioprocess development. Therefore, three main aspects have been studied in this
thesis: 1) characterization of mass transfer in microtiter plates (MTPs), 2) development of a new
online-monitoring technique for detecting the most relevant fermentation parameters, 3) proof of
scalabilty from microtiter plates to stirred tank fermenters. The oxygen mass transfer into microtiter
plates was characterized with the sulfite oxidation method in 24-, 48- and 96-well MTPs. On the one
hand, the results pointed out that the achieved maximum oxygen transfer capacities (OTR ) of 0.039 max
and 0.052 mol/L/h (k a= 250 and 300 1/h) for round 24- and 96-well MTPs could be limiting for most of L
microbial fermentations. On the other hand, round 48-well MTPs provided very high OTR up to 0.28 max
mol/L/h (k a= 1600 1/h) with the drawback that these high values could only be achieved at very high L
shaking frequencies (1400 1/min) and very small filling volumes (300 µL) which were not very suitable
for online monitoring in the microwells and for further offline sample analysis. Therefore, also the 48-
well Flowerplate was characterized in oxygen mass transfer resulting in high OTR values of 0.14 max
mol/L/h (k a= 800 1/h) at acceptable filling volumes (500 µL) for online detection and further offline L
sample analysis. Furthermore, a new online monitoring technique for the online detection of all
relevant fermentation parameters such as biomass and fluorescent protein concentrations as well as
pH and dissolved oxygen tension (DOT) by optodes in continuously shaken MTPs - in the meantime
commercialized under the trade name BioLector - was developed and validated. This technique was
approved in several examples of microbial fermentations with Eschericha coli and the yeast
Hansenula polymorpha as model organisms. It could be demonstrated that online biomass detection is
possible up to biomass concentrations of 50 g/L cell dry weight due to a linear correlation between
scattered light intensities and cell dry weight in the mentioned range. The scalability of microtiter plate
fermentations to standard stirred tank fermenters was proven with Eschericha coli and the yeast
Hansenula polymorpha. A comparison of fermentations in 200 µL in MTPs with fermentations in 1.4 L
stirred tank fermenter depicted the same fermentation kinetics and fermentation times as well as
absolute concentrations proved by online biomass and protein expression of green fluorescent protein
(GFP) as model protein in both scales. The combination of the online monitoring technique with the
recently developed Flowerplate finally demonstrated the broad applicability of this technology in
upstream bioprocessing providing all relevant fermentation parameters online at elevated oxygen
transfer conditions. This technology fulfills all requirements of an ideal microbioreactor system by
featuring high-throughput, high information content and scalability to standard stirred tank fermenters.
The application of this new technology in bioprocess R&D could dramatically facilitate and accelerate
clone screening and fermentation development by providing more relevant bioprocess information at
microscale.

I


II
Preface

This thesis was conducted at the RWTH Aachen University, AVT-Biochemical
Engineering from January 2002 to April 2006 and later completed at the spin-off of
that department, m2p-labs GmbH.

I am grateful to Prof. Dr.-Ing. Jochen Büchs for receiving me back from industry and
giving me the opportunity to conduct my PhD thesis at his department on a very
interesting and powerful topic. I greatly appreciate his willingness for fruitful
discussions on my PhD topic and further themes of new research funding projects.
Furthermore, I want to thank Prof. G. Gellissen for taking over the co-referee of my
PhD work.

Special thanks go to all the students who significantly contributed to this work:
E. Zang, D. Dreymüller, S. Kaseder, K. Luft, B. Hofmann, C. Faulhammer, A. Käser,
Krümmel C. Engelbrecht, A. Daub, J. Höth, L. Wang, D. Ewert, R.-K. Tan, J. Ryll and
A.K. Hillmer.

Further I would like to acknowledge C. Amuel, M. Sack, M. Suckow, M. Piontek, F.
Müller for providing various strains producing fluorescent and recombinant proteins.

I want to thank all members of the AVT-Biochemical Engineering and especially
C. Stöckmann, K. Rachinskiy, C. Ferloni, A. Knoll, C. Peter, K. Raval and C. Müller
for the good and fruitful atmosphere at the department. I greatly appreciated the
contributions of the mechanical (H. Alt and his colleagues) and electrical workshop
(U. Kosfeld and Tom) of the biology faculty and the support from A. Will and G.
Schmeißer in literature search and administration.

Very special thanks I want lend to the co-founders of m2p-labs, Mirko Hoffmann, Ingo
Klammer and Carsten Müller, who finally convinced me to contribute in the
foundation of this company which now sells the outcome of this thesis.


III
Finally, I want to express my gratitude to my friends and family for the continual
support over the last years. Especially, I want to thank Gabriela Grassmann for her
great patience and her continuous motivation to finalize this thesis.

My very last thanks I want to lend to Negar who gave me the motivation for the finish
of this thesis, but sadly she cannot experience my great relief anymore.

Parts of this thesis have already been published:
Kensy F, Zimmermann HF, Knabben I, Anderlei T, Trauthwein H, Dingerdissen U,
Büchs J: Oxygen transfer phenomena in 48-well microtiter plates: determination
by optical monitoring of sulfite oxidation and verification by real-time
measurement during microbial growth. Biotechnol Bioeng 2005, 89(6):698-708.
Kensy F, John GT, Hofmann B, Büchs J: Characterisation of operation conditions
and online monitoring of physiological culture parameters in shaken 24-well
microtiter plates. Bioprocess Biosyst Eng 2005, 28(2):75-81.
Kensy F, Zang E, Faulhammer C, Tan RK, Büchs J: Validation of a high-
throughput fermentation system based on online monitoring of biomass and
fluorescence in continuously shaken microtiter plates. Microb Cell Fact 2009,
8:31.
Kensy F, Engelbrecht C, Büchs J: Scale-up from microtiter plate to laboratory
fermenter: Evaluation by online monitoring techniques of growth and protein
expression in Escherichia coli and Hansenula polymorpha fermentations.
Microb Cell Fact 2009, Microb Cell Fact 2009, 8:68.



Other contributions to publications and patents during this PhD thesis:

Publications:

Hofmann MC, Kensy F, Büchs J, Mokwa W, Schnakenberg U: Transponder-based
sensor for monitoring electrical properties of biological cell solutions. J Biosci
Bioeng, 2005, 100(2), 172-177.

IV

Hofmann MC, Ellersiek D, Kensy F, Büchs J, Mokwa W, Schnakenberg U: Galvanic
decoupled sensor for monitoring biomass concentration during fermentation
processes. Sens Actuator B-Chem 2005, 111:370-375.
Funke M, Diederichs S, Kensy F, Müller C, Büchs J: The baffled microtiter plate:
increased oxygen transfer and improved online monitoring in small scale
fermentations. Biotechnol Bioeng 2009, 103(6):1118-1128.
Huber R, Ritter D, Hering T, Hillmer AK, Kensy F, Müller C, Wang L, Büchs J:
Robo-Lector – a novel platform for automated high-throughput cultivations in
microtiter plates with high information content. Microb Cell Fact 2009, 8:42.


Patents:

Funke M, Kensy F, Müller C, Büchs J: Mikroreaktor (Flowerplate)
German patent application DE 102008 008 256 A1, Priority: 08.10.2007.

Büchs J, Mokwa W, Schnakenberg U, Kensy F, Müller C, Klammer I, Buchenauer A,
Hofmann M: Vorrichtung und Verfahren zur Zu- und Abfuhr von Fluiden in
geschüttelten Mikroreaktoren Arrays. German patent application DE 10 2006 030
068 A1, Priority: 28.06.2006.

Büchs J, Kensy F, Samorski M: Verfahren und Vorrichtung zur Erfassung von
Prozeßparametern von Reaktionsflüssigkeiten in mehreren geschüttelten
Mikroreaktoren. Granted European patent EP1730494B1, Priority: 02.04.2004

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