High-throughput screening of optimal process conditions using model predictive control.

Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g., fed-batch or continuous) and generate significant amounts of measurement data. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, to name a few. In this study we implement a model predictive control (MPC) framework to demonstrate: (1) the challenges in terms of mathematical model structure, state, and parameter estimation, and optimization under highly nonlinear and stiff dynamics in biological systems, (2) the adaptations required to enable the application of MPC in high throughput bioprocess development, and (3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the dissolved oxygen tension profile.

A Unique Response Behavior in the Dissolved Oxygen Tension in E. coli Minibioreactor Cultivations with Intermittent Feeding.

Intermittent bolus feeding for E. coli cultivations in minibioreactor systems (MBRs) profoundly affects the cell metabolism. Bolus feeding leads to temporal substrate surplus and transient oxygen limitation, which triggers the formation of inhibitory byproducts. Due to the high oxygen demand right after the injection of the substrate, the dissolved oxygen tension (DOT) signal exhibits a negative pulse. This contribution describes and analyzes this DOT response in E. coli minibioreactor cultivations. In addition to gaining information on culture conditions, a unique response behavior in the DOT signal was observed in the analysis. This response appeared only at a dilution ratio per biomass unit higher than a certain threshold. The analysis highlights a plausible relationship between a metabolic adaptation behavior and the newly observed DOT signal segment not reported in the literature. A hypothesis that links particular DOT segments to specific metabolic states is proposed. The quantitative analysis and mechanistic model simulations support this hypothesis and show the possibility of obtaining cell physiological and growth parameters from the DOT signal.

Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations.

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 in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. 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-batch cultivations were run under the desired conditions, generating sufficient information to define the fastest-growing strain in an environment with oscillating glucose concentrations similar to industrial-scale bioreactors.

Monte Carlo Simulations for the Analysis of Non-linear Parameter Confidence Intervals in Optimal Experimental Design.

Especially in biomanufacturing, methods to design optimal experiments are a valuable technique to fully exploit the potential of the emerging technical possibilities that are driving experimental miniaturization and parallelization. The general objective is to reduce the experimental effort while maximizing the information content of an experiment, speeding up knowledge gain in R&D. The approach of model-based design of experiments (known as MBDoE) utilizes the information of an underlying mathematical model describing the system of interest. A common method to predict the accuracy of the parameter estimates uses the Fisher information matrix to approximate the 90% confidence intervals of the estimates. However, for highly non-linear models, this method might lead to wrong conclusions. In such cases, Monte Carlo sampling gives a more accurate insight into the parameter's estimate probability distribution and should be exploited to assess the reliability of the approximations made through the Fisher information matrix. We first introduce the model-based optimal experimental design for parameter estimation including parameter identification and validation by means of a simple non-linear Michaelis-Menten kinetic and show why Monte Carlo simulations give a more accurate depiction of the parameter uncertainty. Secondly, we propose a very robust and simple method to find optimal experimental designs using Monte Carlo simulations. Although computational expensive, the method is easy to implement and parallelize. This article focuses on practical examples of bioprocess engineering but is generally applicable in other fields.

Model-based identifiable parameter determination applied to a simultaneous saccharification and fermentation process model for bio-ethanol production.

In this work, a methodology for the model-based identifiable parameter determination (MBIPD) is presented. This systematic approach is proposed to be used for structure and parameter identification of nonlinear models of biological reaction networks. Usually, this kind of problems are over-parameterized with large correlations between parameters. Hence, the related inverse problems for parameter determination and analysis are mathematically ill-posed and numerically difficult to solve. The proposed MBIPD methodology comprises several tasks: (i) model selection, (ii) tracking of an adequate initial guess, and (iii) an iterative parameter estimation step which includes an identifiable parameter subset selection (SsS) algorithm and accuracy analysis of the estimated parameters. The SsS algorithm is based on the analysis of the sensitivity matrix by rank revealing factorization methods. Using this, a reduction of the parameter search space to a reasonable subset, which can be reliably and efficiently estimated from available measurements, is achieved. The simultaneous saccharification and fermentation (SSF) process for bio-ethanol production from cellulosic material is used as case study for testing the methodology. The successful application of MBIPD to the SSF process demonstrates a relatively large reduction in the identified parameter space. It is shown by a cross-validation that using the identified parameters (even though the reduction of the search space), the model is still able to predict the experimental data properly. Moreover, it is shown that the model is easily and efficiently adapted to new process conditions by solving reduced and well conditioned problems.

Optimal determination of steric mass action model parameters for beta-lactoglobulin using static batch experiments.

In this work, parameters of the steric mass-formalism SMA are optimally ascertained for a reliable determination of the adsorption isotherms of beta-lactoglobulin A and B under non-isocratic conditions. For this purpose, static batch experiments are used in contrast to the protocols based on different experimental steps, which use a chromatographic column. It is shown that parameters can already be determined for a small number of experiments by using a systematic procedure based on optimal model-based experimental design and an efficient NLP-solver. The in different works observed anti-Langmuir shape of the isotherm for small concentrations of beta-lactoglobulin A was corroborated. Moreover, we also found indications for a porosity variation with changing protein concentrations.