Online monitoring in continuously shaken microtiter plates for scalable upstream bioprocessing

  • Online-Monitoring in kontinuierlich geschüttelten Mikrotiterplatten für skalierbare Ergebnisse in der Fermentationsentwicklung

Kensy, Frank Torsten; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2011)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2010


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 (OTRmax) of 0.039 and 0.052 mol/L/h (kLa= 250 and 300 1/h) for round 24- and 96-well MTPs could be limiting for most of microbial fermentations. On the other hand, round 48-well MTPs provided very high OTRmax up to 0.28 mol/L/h (kLa= 1600 1/h) with the drawback that these high values could only be achieved at very high 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 OTRmax values of 0.14 mol/L/h (kLa= 800 1/h) at acceptable filling volumes (500 µL) for online detection and further offline 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.