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March 15, 2010 (Vol. 30, No. 6)

High-Throughput Shaken Microbioreactors

Methodologies to Simplify and Automate Bioprocess Characterization

  • It is well known that upstream bioprocessing is sensitive and prone to errors. As a result, an increasing number of new processes are being developed to produce biopharmaceuticals, enzymes, or biofuels more efficiently. In addition, researchers are exploring the process and attempting to gain a better understanding of the implications of programs such as PAT and QbD.

    Current fermentation technologies, e.g., stirred tank fermentors, no longer meet the needs of the industry. In the past several years, there has been tremendous interest in microbioreactors as an alternative technology for accelerating bioprocess development. Shaken microtiter plates (MTPs) have garnered a lot of attention for their simplicity and high-throughput. Microtiter plates are as common and easy as shake flasks, yet they provide a high-throughput capacity and are compatible with automation.

    Standard microplate formats such as 24- or 96-well plates were not developed for fermentation applications and, therefore, they provide only limited oxygen supply for aerobic fermentations. A solution is provided with the 48-well Flowerplate from m2p-labs in which well geometries have been optimized (Figure 1).

    This new microplate with flower-shaped wells provides microbial cells with oxygen transfer rates (OTRs) of up to 0.2 mol/L/h, which is equivalent to a specific mass transfer coefficient kLa of 1,140 h-1. For cell culture applications, these high oxygen transfer rates are not necessary due to the slower cell metabolism, therefore, the round 48-well cell culture plates have smaller kLa values in the range of 10 to 100 h-1.

    BioLector is an online measurement technique for continuously shaken microtiter plates. It was developed in the department of biochemical engineering at RWTH Aachen University by a group headed by Professor Jochen Büchs.

  • Click Image To Enlarge +
    Figure 1. Flowerplate with the BioLector measurement principle

    All relevant fermentation parameters such as biomass concentration (via scattered light), pH, DOT, and even fluorescent proteins can be detected online in each well during the orbital shaking process (Figure 1). The continuous shaking ensures that mixing and oxygen supply are steady, thus avoiding measurement artifacts. Microplates are covered with gas-permeable membranes for monoseptical operation of the cultures.

    The BioLector can incubate one microplate in its incubation chamber where temperature (20–50ºC), humidity (>75% rH), and the gas atmosphere (O2: 0–21% and CO2: 0–10%) can be controlled. For higher throughput, the BioLector can be used just as a reading station, and microplates can be incubated on a separate incubation shaker at the same culture conditions as in the BioLector. The Flowerplate and a cell culture plate can be used with common MTP holders.

  • Scale Up

    Click Image To Enlarge +
    Figure 2. Comparison of parallel E. coli fermentations in a microtiter plate and stirred tank fermentor

    When working with a high-throughput platform at microscale it is important that  the results received from microscale can easily be transferred to laboratory-scale. If the scale-up is validated, the high-throughput experimentation platform can facilitate bioprocess development allowing more process-characterization tasks to be scaled down to microscale. To confirm the scalability of microfermentation in the BioLector to a laboratory fermentor, parallel fermentations of E. coli and the yeast Hansenula polymorpha were conducted in both scales, with 200 µL and 1.4 L respectively.

    For both scales, mass transfer conditions (kLa values) were applied, which ensured no significant oxygen limitation. Figure 2 depicts the biomass (A), protein-expression development (B), and the process parameters (C) over time. Both the biomass and the protein expression (with GFP as a model protein) curves of all induced cultures (II–IV), showed almost identical behavior in both scales.

    From these results it can be concluded that, in respect to biomass and protein-expression development, which are the basic evaluation criteria of a fermentation process, these microbial fermentations are scalable from microtiter plates to laboratory fermentors.

    As a negative control, a noninduced E. coli culture was cultivated in parallel to the induced cultures. Figure 2 shows how large the differences can be when applying different culture conditions. These results were also confirmed by fermentations with Hansenula polymorpha.

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