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

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.

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.

Figure 1. Flowerplate with the BioLector measurement principle

Scale Up

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.

Figure 2. Comparison of parallel E. coli fermentations in a microtiter plate and stirred tank fermentor

Automation of Upstream Bioprocessing

Another important aspect of microfermentations is automation. While the BioLector provides most of the fermentation parameters online, detecting target proteins or substrate concentrations online is not always possible. Therefore, it is useful to combine the BioLector with an automated liquid-handling platform.

The Robolector includes pipetting robots and the BioLector (Figure 3).  RoboLector software provides signal-triggered manipulation of the fermentation process in the BioLector.

At a predefined set point, e.g., a certain biomass concentration or DOT level, the system is able to add inducers or substrates to each individual well or to take samples out of them. Sampled fermentation broths can immediately be cooled down to -10ºC in a cooling station placed on the liquid-handling platform. The system can also further process the samples or disrupted cells in a centrifuge to recover the supernatant for further downstream processing or analysis.

The RoboLector platform was recently evaluated in an induction-profiling experiment with E. coli in which inducer concentrations of IPTG and induction times were varied. The results showed that maximum space-time yields could be achieved at low inducer concentrations of 50 mM IPTG and induction times between 3.5 to 4.0 hours.

The BioLector facilitates high-throughput fermentations with online monitoring of all relevant fermentation parameters in shaken microtiter plates. The development of new flower-shaped well geometries, realized in the Flowerplate, provide unlimited oxygen transfer conditions (OTR up to 0.2 mol/L/h) for most microbial fermentation applications, thus, avoiding the need for active pH control due to the production of organic acids under micro-aerobic conditions.

RoboLector allows for the automation  of upstream bioprocessing with standard liquid-handling systems. In addition, its use makes it possible for fermentations to be conducted automatically in a high-throughput screening environment.

Figure 3. RoboLector platform for automated upstream bioprocessing

Frank Kensy ([email protected]) is managing director of m2p-labs.

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