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The calculation of metabolic turnover rates is essential for the scalable design, analysis and control of bioprocesses. A novel rate calculation algorithm is presented that is based on the dynamic adaptation of window sizes in order to deliver robust and precise rates with uniform signal-to-noise ratios. Additionally a model-based generic algorithm for deriving optimal rate calculation workflows was developed. The generic algorithms delivered more precise and accurate rates for online and offline signals, which was demonstrated for both in silico- and real batch and fed-batch fermentation process data. The presented algorithms will strongly support bioprocess development and control as enabling tools for multivariate data analysis, mechanistic modeling and dynamic experimentation.

Identification of critical process parameters that impact product quality is a central task during regulatory requested process validation. Commonly, this is done via design of experiments and identification of parameters significantly impacting product quality (rejection of the null hypothesis that the effect equals 0). However, parameters which show a large uncertainty and might result in an undesirable product quality limit critical to the product, may be missed. This might occur during the evaluation of experiments since residual/un-modelled variance in the experiments is larger than expected a priori. Estimation of such a risk is the task of the presented novel retrospective power analysis permutation test. This is evaluated using a data set for two unit operations established during characterization of a biopharmaceutical process in industry. The results show that, for one unit operation, the observed variance in the experiments is much larger than expected a priori, resulting in low power levels for all non-significant parameters. Moreover, we present a workflow of how to mitigate the risk associated with overlooked parameter effects. This enables a statistically sound identification of critical process parameters. The developed workflow will substantially support industry in delivering constant product quality, reduce process variance and increase patient safety.

During the regulatory requested process validation of pharmaceutical manufacturing processes, companies aim to identify, control, and continuously monitor process variation and its impact on critical quality attributes (CQAs) of the final product. It is difficult to directly connect the impact of single process parameters (PPs) to final product CQAs, especially in biopharmaceutical process development and production, where multiple unit operations are stacked together and interact with each other. Therefore, we want to present the application of Monte Carlo (MC) simulation using an integrated process model (IPM) that enables estimation of process capability even in early stages of process validation. Once the IPM is established, its capability in risk and criticality assessment is furthermore demonstrated. IPMs can be used to enable holistic production control strategies that take interactions of process parameters of multiple unit operations into account. Moreover, IPMs can be trained with development data, refined with qualification runs, and maintained with routine manufacturing data which underlines the lifecycle concept. These applications will be shown by means of a process characterization study recently conducted at a world-leading contract manufacturing organization (CMO). The new IPM methodology therefore allows anticipation of out of specification (OOS) events, identify critical process parameters, and take risk-based decisions on counteractions that increase process robustness and decrease the likelihood of OOS events.

In biopharmaceutical process development and manufacturing, the online measurement of biomass and derived specific turnover rates is a central task to physiologically monitor and control the process. However, hard-type sensors such as dielectric spectroscopy, broth fluorescence, or permittivity measurement harbor various disadvantages. Therefore, soft-sensors, which use measurements of the off-gas stream and substrate feed to reconcile turnover rates and provide an online estimate of the biomass formation, are smart alternatives. For the reconciliation procedure, mass and energy balances are used together with accuracy estimations of measured conversion rates, which were so far arbitrarily chosen and static over the entire process. In this contribution, we present a novel strategy within the soft-sensor framework (named adaptive soft-sensor) to propagate uncertainties from measurements to conversion rates and demonstrate the benefits: For industrially relevant conditions, hereby the error of the resulting estimated biomass formation rate and specific substrate consumption rate could be decreased by 43 and 64 %, respectively, compared to traditional soft-sensor approaches. Moreover, we present a generic workflow to determine the required raw signal accuracy to obtain predefined accuracies of soft-sensor estimations. Thereby, appropriate measurement devices and maintenance intervals can be selected. Furthermore, using this workflow, we demonstrate that the estimation accuracy of the soft-sensor can be additionally and substantially increased. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9711-9) contains supplementary material, which is available to authorized users.

Background Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. Results We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through “random-walk” lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. Conclusions Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0463-8) contains supplementary material, which is available to authorized users.