Insufficient mixing in large-scale bioreactors, provokes gradient zones of substrate, dissolved oxygen, pH and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and -methyl-norleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids, but also that pyruvate pulses produce the same effect. Therefore we combined the enzyme based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions, produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor. To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance of the selected cell factory in larger reactors, has a major influence on the performance of the final process. To overcome this, scaledown approaches are essential to run screenings that show the real cell factory performance at industrial like conditions. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batches ran under the desired conditions generating sufficient information to define the fastest growing strain in an environment with varying glucose concentrations similar to industrial scale bioreactors.

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations have been used to accelerate and optimize bioprocess development. As implementation of fed-batch conditions, multiple options of process control and sample analysis are possible, these systems represent valuable screening tools for large-scale production. However, the dynamic behavior of cultivations has not yet been considered regarding data evaluation and decision making during high-throughput screening in mini-bioreactors. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed in 48 parallel fed-batch cultivations regarding 16 experimental conditions. Automated parallel process control, frequent sampling and analysis were implemented. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable and suitable for automatized process development reducing the experimental times and costs.