Design of Experiment (DoE) for Fed-batch Process Development in Shaken Cultures

Determination of reproducibility and scalability in Escherichia coli cultures grown under fed-batch conditions using SensorDishes® and Sensor Flasks

F. Glauche, N. Cruz, A. Knepper, M. Föllmer, S. Junne, P. Neubauer
Laboratory of Bioprocess Engineering, Department of Biotechnology, TU Berlin, Germany

When designing a recombinant protein production process, a high number of parallel cultivations need to be carried out. This is mostly performed using batch cultures in shake flasks or micro well plates, where the fermentation conditions are not monitored. To overcome this limitation, the SensorDish® Reader and Shake Flask Reader were combined with the enzymatic glucose delivery system (EnBase®) for Escherichia coli cultivations. SensorDish® cultures were found to yield reproducible process data and the results were directly comparable to the shake flask scale.

Especially in the biotech industry, a consistent process development from microliter to industrial scale requires the consideration of many different perspectives in order to achieve a complete understanding of the transition process. In this study a DoE was created to determine optimal parameters at two different cultivation scales. Such batch-type cultivation principles in orbitally shaken systems (mostly shake flasks, but also micro well plates and bench-scale fermenters) are used to obtain acceptable results with as less effort as possible. Unfortunately, this is mostly carried out without considering the important influence of the conditions in large-scale production on the quality of the product and the yield of the process. The major parameters, which are overseen in screening and laboratory scale are: 1) the fed-batch operation, where the dosing of carbon source and the pH are controlled, and 2) the concentration gradients, caused by large volumes, insufficient stirring / mixing capacity, and operation cost reduction. Batch processes at laboratory scale have high initial concentrations of carbon source and the pH may change over time. Furthermore, shake flask cultivations present insignificant concentration gradients in the medium hindering the study of bacterial growth under rapid changing conditions (e. g. glucose pulses). With micro well plates only little information is generated and the obstacles mentioned above are also present. In addition, sampling as well as high precision analysis is limited due to the small volumes. It is therefore important to understand the real limitation of each scale (micro well, flask, minireactor, large scale reactor), as well as its relation to all other steps in the product development chain. Furthermore, it is essential to create reliable mathematical, analytic, monitoring and control methods to transmit the information between the different stages. In this work a comparison of fed-batch cultivations in two different scales, namely 24-well plates (3.3 mL) and shake flasks (125 mL) are compared.  To achieve this, the PreSens fluorescent sensor technology is used to allow the online monitoring of both dissolved oxygen and pH in the culture (Fig. 1). By these means, a mathematical relation between the micro- and milliliter scales is to be developed. This approach should enable a better design of shake flask experiments, based on previous information obtained in well plate scale.

Materials & Methods

In order to perform substrate limited fed-batch cultivations in plates and flasks, the enzymatic glucose delivery system EnBase® (BioSilta) was used. EnBase® emulates glucose feed releasing glucose from a polysaccharide by means of an enzymatic reaction. The feed of glucose in the culture can be controlled with the amount of the enzyme EnZ I´m added. Goal of the experiments was to determine optimal volume and enzyme concentration for plates and flasks. A D-optimal design of experiment was created with MODDE (Umetrics), with the factors enzyme (EnZ I´m) concentration (0 to 30 U/L) and volume (0.5 to 2.1 mL in 24-well plates, 7 to 15 mL in flasks). Optical density was chosen as response. 11 tests were run at varying volumes and enzyme concentrations for the two different scales: 24-well OxoDishes® and HydroDishes® with the SDR SensorDish® Reader measuring system, and shake flasks equipped with pH and DO fluorescent sensors and measured by the SFR Shake Flask Reader. The SensorDishes® were combined with `System Duetz´ sandwich covers and clamp systems (EnzyScreen). In addition to the online measurements of pH and DO, bacterial growth was measured in a spectrophotometer at 600 nm (OD600). The enzymatic feed was carried out with EnPresso® medium (BioSilta) prepared according to the manufacturer´s description and inoculated with E. coli BL21 cells; the initial OD600 of the cultures was between 0.1 and 0.3. The pH and DO were continuously monitored in the micro well plates and in the shake flasks during the whole cultivation to ensure constant conditions without oxygen limitation and a pH level between 5 and 7.5. The overnight cultures were removed from the respective readers at two-hour intervals to determine the optical density OD600.

Comparison of SDR and SFR

As depicted in Fig. 2, all dissolved oxygen curves present the characteristic trends of a substrate limited fed-batch culture. The concentration of enzyme is constant throughout each experiment; this induces a quasi-constant feed of glucose during the cultivation. In the first phase, the low cell density results in a reduced total glucose uptake capacity allowing for glucose accumulation, causing an initial phase of exponential growth. The rapid increase of the respiration capacity of the bacteria is indicated by the accelerated drop of the DOT curve. The decrease in the pH values indicates secretion of acidic compounds, mainly acetic acid. The second phase is characterized by substrate limitation. Excess glucose is consumed and cells are obliged to decrease the growth rate and respiration to adapt to the new conditions. As shown in Fig. 2, the initial decrease of DOT recorded in the SDR, as well as in the SFR is dependent on the amount of glucose-releasing enzyme added to the culture. Therefore, an optimal amount of enzyme for rapid aerobic growth can be determined (Fig. 2 A - C). As expected, cultures in the shake flask allow a better oxygen transfer so that enzyme concentrations can be increased up to 23.3 U L-1 without reaching oxygen limitation. The trends of the SDR experiments were found to be reproducible in the shake flask system; a similar DOT and pH profile was recorded (Fig. 2 D - F). Furthermore, reproducibility of the cultures was demonstrated. Variations between different wells and different cultures were found to be statistically insignificant (data not shown).


DOT and pH are essential measurements for monitoring the state of cultures in bio production. The possibility to monitor both, DOT and pH, already in early development stages like screening experiments in micro well plates and shake flasks, is a huge step towards faster bioprocess development. Combining these new measuring equipments with the adequate models for monitoring and scale up, permits a more efficient information flow between small and medium scale. Screening efficiency is increased by enabling cellular growth under aerobic, substrate-limited and pH stabilizing conditions. Controlled fed-batch cultivations allow not only higher product formation, but also a higher similarity to large scale production conditions.


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