Minimal time control of fed-batch processes with growth functions having several maxima

We address the issue of minimal time optimal control of fedbatch reactor in presence of complex non monotonic kinetics, that can be typically characterized by the combination of two Haldane models. The optimal synthesis may present several singular arcs. Global optimal trajectory results are provided on the basis of a numerical approach that considers an approximation method with smooth control inputs

Optimal feedback synthesis and minimal time function for the bioremediation of water resources with two patches

This paper studies the bioremediation, in minimal time, of a water resource or reservoir using a single continuous bioreactor. The bioreactor is connected to two pumps, at different locations in the reservoir, that pump polluted water and inject back sufficiently clean water with the same flow rate. This leads to a minimal-time optimal control problem where the control variables are related to the inflow rates of both pumps. We obtain a non-convex problem for which it is not possible to directly prove the existence of its solutions. We overcome this difficulty and fully solve the studied problem by applying Pontryagin's principle to the associated generalized control problem. We also obtain explicit bounds on its value function via Hamilton-Jacobi-Bellman techniques

Bioprocess Systems Engineering Applications in Pharmaceutical Manufacturing

Biopharmaceutical and pharmaceutical manufacturing are strongly influenced by the process analytical technology initiative (PAT) and quality by design (QbD) methodologies, which are designed to enhance the understanding of more integrated processes. The major aim of this effort can be summarized as developing a mechanistic understanding of a wide range of process steps, including the development of technologies to perform online measurements and real-time control and optimization. Furthermore, minimization of the number of empirical experiments and the model-assisted exploration of the process design space are targeted. Even if tremendous progress has been achieved so far, there is still work to be carried out in order to realize the full potential of the process systems engineering toolbox. Within this reprint, an overview of cutting-edge developments of process systems engineering for biopharmaceutical and pharmaceutical manufacturing processes is given, including model-based process design, Digital Twins, computer-aided process understanding, process development and optimization, and monitoring and control of bioprocesses. The biopharmaceutical processes addressed focus on the manufacturing of biopharmaceuticals, mainly by Chinese hamster ovary (CHO) cells, as well as adeno-associated virus production and generation of cell spheroids for cell therapies

Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells

Krattenmacher F. Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Bielefeld: Universität Bielefeld; 2020.Chemically defined media (CDM) for cell culture are routinely used in industrial processes for recombinant protein production from mammalian expression systems as for example Chinese hamster ovary (CHO) cells. As CDM are nowadays considered as the industry standard the focus has shifted from implementation and improvement of performance to additionally their chemical behavior and the impact on process robustness. Since CDM are highly concentrated aqueous mixtures of versatile chemical compounds one particular problem in this context is the high risk for chemical reactions and instability. Therefore, a major focus of this thesis is the generation of understanding for chemical interactions of CDM compounds and especially the establishment of analytical technologies for the purpose of media characterization. Thus, a mixed mode liquid chromatography tandem mass spectrometry (LC-QqQ-MS) method that is able to simultaneously quantify the majority of media compounds has been developed and validated. This powerful method has been applied to characterize the chemical behavior of feed media under process relevant conditions as preparation and storage. Further on line and off line analytics have been applied to gain insight into CDM chemistry. The application of probes measuring standard parameters have shown the dynamic behavior of chemical key parameters during CDM powder hydration. A Particle probe, such as the focused beam reflectance measurement (FBRM), has been shown to be useful for dissolution behavior investigations of different media recipes or powder compositions. However, it is rather difficult to establish the technology for batch to batch comparison or the monitoring of deviations from the standard preparation conditions. Media preparations with simplified media powders revealed that the compounds ascorbic acid and phosphates cause an apparent drop in dissolved oxygen concentration upon iron compound addition. The combination of the experiments with the newly developed LC QqQ MS method confirmed the comparability of chemical behavior in different media matrixes of most of the CDM compounds but highlighted some differences. Furthermore, measurements with the LC-QqQ-MS showed that the effect of preparation temperature and relevant storage conditions on media stability were negligible. In contrast, measurement of samples over storage time identified unstable compounds. A closer look at the media after storage showed that some formulations formed precipitate during storage and the collection of the solid material on filter membranes revealed their different appearance. Investigations of the material with specialized analytics proved that their identity was heterogeneous. One precipitate that was drawing attention on itself was of silver color and could be shown to consist of Sulphur

Processes controlling the quantities of biogenic materials in lakes and reservoirs subject to cultural eutrophication

The processes which control the growth, composition, succession and loss from suspension of phytoplankton algae are briefly reviewed, with special reference to function in eutrophic reservoir systems. The ecology of larger algal biomasses supported by high nutrient loading rates are more likely to be subject to physical (wash-out, underwater light penetration, thermal stability and mixing) than to chemical constraints. Sudden changes in the interactions between physical factors temporarily impair the growth of dominant algal species, and advance the succession. Certain algae may be cropped heavily, but selectively, by zooplankton feeding, but they are rarely the species which cause problems in waterworks practice. Grazing, however, does influence succession. A deeper understanding of the operation of loss control mechanism is urgently required. Potentially, manipulation of the physical environment provides an important means of alleviating day-to-day algal problems in eutrophic reservoirs; in terms of cost effectiveness these may prove to be more attractive than reducing nutrient loads at source

Tools for improving feeding strategies in a SBR with several species

International audienceThis paper analyzes feeding strategies in a sequential batch reactor (SBR) with the objective of reaching a given (low) substrate level as quickly as possible for a given volume of water. Inside the SBR, several species compete for a single substrate, which leads to a minimal time control problem in which the control variable is the feeding rate. Following [10], we allow the control variable to be a bounded measurable function of time combined with possible impulses associated with instantaneous dilutions. For this problem, the ex-tremal trajectories of the singular arc type are characterized as the strategies used to maintain the substrate at a constant level. Because this optimization problem is difficult to solve, this characterization provides a valuable tool for investigating the optimality of various feeding strategies. Our aim is thus to illustrate the use of this tool by proposing potential optimal feeding strate-gies, which may then be compared with other more intuitive strategies. This aim was accomplished via several numerical experiments in which two specific strategies are compared

Strategies of plasmid DNA production and their influence on therapeutic applications

Current developments in gene therapy and DNA vaccination, plasmid DNA vectors are becoming increasingly appealing as therapeutics towards a large number of diseases such as cancer, infectious and cardiovascular diseases. This popularity is creating the demand for high quantities of highly purified plasmid DNA which in turn requires the design of high pDNA yield bioprocesses. However, opposed to recombinant protein production, research on pDNA production is still needed, in order to have a clear comprehension of all the challenges and bottlenecks faced during the production of plasmid DNA (pDNA). The design of a plasmid DNA production process usually begins with the choice of a suitable culture medium to cultivate the expression system containing the therapeutic plasmid. After defining all medium components, the influence of culture conditions, such as pH, temperature and dissolved oxygen, on biomass and plasmid yields is generally studied. Since the appropriate conditions for maximizing biomass production and plasmid replication are not usually the same, a compromise solution is usually considered in these cases. When designing a large scale plasmid DNA production process, the employment of a correct fermentation strategy is also necessary in order to improve yields while reducing production costs. So far, reports on plasmid DNA production in E. coli are focused essentially on the influence of different medium composition, fermentation conditions and feeding strategies on overall biomass and pDNA mass and volumetric titres. Nevertheless, due to the complex nature of microbial growth and the application of several modes of operation such as batch, fed-batch and continuous cultivations, the constant monitoring and control of pDNA bioprocesses represents an engineering challenge that should not be disregarded. As a highest yield process may not correspond necessarily to the best fermentation design, the improvement in off-line, at-line and online monitoring techniques should be seen as a crucial task in the design of the fermentation. Two of the most relevant factors for fermentation performance are the existence of host cell metabolic stress and plasmid instability; hence, the characterization of cell physiology and plasmid segregational stability has to be considered and monitored during the process. With this thesis, we attempted to improve plasmid DNA yields while gaining new insights on plasmid DNA fermentation processes through the use of novel monitoring techniques such as flow cytometry and real-time quantitative PCR. We started this work studying the influence of growth temperature and tryptone concentration on plasmid DNA production in a previously developed semi-defined medium. The analysis of pDNA yields and E. coli morphology revealed that higher pDNA specific yields were obtained at higher temperatures (37 and 40 ºC). Also, at these temperatures, E. coli filamentation was observed, possibly indicating a higher metabolic stress due to higher plasmid replication and higher culture temperatures. When analyzing the influence of tryptone concentration on plasmid yield, the best results were achieved with the lowest tryptone concentration used. The use of limiting tryptone concentrations at a temperature of 37 ºC was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure (supercoiled isoform); thus favouring the attainment of product quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA amplification was improved, proving that this strategy is able to increase pDNA yield even at small scale. Since this first study revealed some evidence of E. coli metabolic stress during cultivation, the next task consisted in the development of new flow cytometric methodologies that allowed single cell physiology monitoring during cultivation for a better understanding of cultivation conditions influence in the host metabolic activity. Because one of the parameters that enable a better characterization of cell metabolic cell as well as population heterogeneity is cell cycle progression, in the second part of the work, we developed a flow cytometric method to evaluate cell cycle progression in E. coli cultivation using a newly developed far-red dye, DRAQ5. In this study we demonstrated that the use of DRAQ5 as a DNA-specific labelling stain provided an easy assessment of intracellular DNA content and cell-cycle phases in the Gram-negative bacteria E. coli. Besides the previously reported method for cell cycle analysis, another method for assessing cell viability was implemented using flow cytometry. In this method, a propidium iodide/bis- (1,3-dibutylbarbituric acid)trimethine oxonol (BOX) dual staining was used to distinguish between three different populations: healthy cells (no staining), cells with depolarized membranes (stained with BOX) and cells with permeabilized membranes (stained with propidium iodide and BOX). In order to evaluate plasmid copy number (PCN) throughout the fermentation, a real-time quantitative PCR method was developed for absolute plasmid copy number quantification in whole E. coli cells. After developing and implementing these methods, we evaluated the impact of several plasmid DNA induction strategies, such as amino acid limitation, AMP addition and temperature up-shift, on cell physiology and plasmid segregational stability. This study showed that all induction strategies caused cell filamentation, due to an increase in forward scatter values, and decreased viability at the end of fermentation, as was seen by an increase in the percentage of depolarized and permeabilized cells. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields and, concomitantly, highest PCN values. In conclusion, amino acid limitation-based amplification strategies seemed to be suitable approaches to be implemented at a large scale level since they do not require any additional energy and also had proved to be efficient in plasmid amplification, without causing any detrimental effects in plasmid stability and cellular viability. The last step of this work aimed at improving plasmid DNA yield through the study of different batch and fed-batch fermentations in bioreactors. Also, the influence of different glycerol and tryptone concentrations and different non-feedback feeding profiles, namely exponential and constant feed rates, on cell physiology and plasmid stability was evaluated by means of flow cytometry and real-time qPCR, respectively; investigating the potential of these two techniques as valuable tools for bioprocess monitoring and design. The results showed that all fermentation strategies caused a slight decrease of cell viability at the end of fermentation, being this decrease more pronounced in fed-batch fermentations than in batch fermentations. The time-course assessment of plasmid copy number revealed that PCN values suffered an increase at the end of batch fermentations, which is in agreement with our previous results obtained in batch fermentations performed in shake flasks. However, in fedbatch fermentations, there were pronounced fluctuations in PCN values throughout the fermentations, indicating some plasmid segregational instability. As supposed, fed-batch fermentations with exponential or constant feeding profiles yielded higher biomass and plasmid DNA than batch fermentations with the highest biomass and plasmid yields being obtained with a fed-batch strategy with an exponential feed rate of 0.2 h-1. Notwithstanding the high biomass (95.64 OD600) and plasmid yields (344.30 mg pDNA/L) obtained, this fermentation also exhibited higher plasmid instability and lower percentage of viable cells. This work showed that the fermentation strategy used, not only influences product yield, but also cell physiology and pDNA segregational stability. Furthermore, the new findings described herein draw attention towards the relevance of monitoring bioprocess performance and not just overall biomass and product yields. In conclusion, in this thesis we evaluated and improved pVAX1-LacZ plasmid production in Escherichia coli DH5 alpha taking into account not only the overall biomass and plasmid yields, but also considering that cell physiology and plasmid segregational stability, that are two pivotal features to the design and development of these production bioprocesses. In order to study these two factors, several techniques were implemented and were later used to evaluate the influence of several fermentation parameters such as induction strategies and fermentation strategies on overall process performance.Work financed by the Portuguese Foundation for Science and Technology (SFRH/BD/41521/2007) under the programe QREN - POPH - Type 4.1 –Advanced Training, cofunded by the European Social Fund and by national funds from the MCTES

Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product

Exosomes are an emerging sub class of extracellular vesicle which are rapidly gaining momentum as a novel therapeutic platform. Their regenerative and therapeutic potential is reflective of the plethora of cell types from which they can be derived. However, as the technology is in its nascent stage, relatively little exists in terms of defined manufacturing processes. As exosome technologies progress towards pre-clinical and clinical stages, the constraints in manufacturing processes, specifically in downstream processing, will need to be overcome. The current “gold standard” for exosome recovery from conditioned culture medium is ultracentrifugation. This step is time consuming, prone to operator error and difficult to scale and translate. The work presented here shows that monolith chromatography is a scalable, and reproducible purification option which can be used to successfully recover functionally active, and highly pure exosomes. Exosomes derived from the clinically relevant stem cell line, CTX0E03, were shown to present the biomarkers CD 9, CD 63 and CD 81, commonly conserved amongst exosome species throughout the literature. The vesicles were characterised as having a size distribution between 20 to 150 nm, and a flotation density between 1.136 – 1.185 g mL-1, as expected based on literature values. Furthermore, exosomes recovered by tangential flow filtration (TFF), were shown to promote fibroblast migration and wound closure (98% ± 1.5%) in an in vitro potency model, in a dose dependant fashion. In contrast exosomes purified by ultracentrifugation could not achieve wound closure, with no significant difference observed over the 72 hour period. TFF recovered exosomes were purified by the processes developed in this thesis. In the first instance they were purified by use of an anion exchange monolith using the quaternary amine ligand. Exosomes were shown to elute broadly over the elution gradient and overlap with DNA and albumin co-present within the feed material. Samples obtained post purification had purity ratios of 1.5 x 10^9 particles per µg of protein and 9.3 x 10^11 particles per µg of DNA impurity. Based on a hypothetical dose size of 10^9 particles per mL this result indicated purities within the WHO guidelines for injectable therapeutics (100 µg of protein, 10 ng DNA per dose) and benchmarked a potential method for purification of exosomes. A second monolith was also tested, using an orthogonal chemistry: hydrophobic interaction with an OH ligand. The results of this column surpassed those of the AEX process and showed a binding affinity beyond the hypothesized values. Unlike the AEX column, the HIC operation did not co-bind impurities in the form of albumin, DNA or even cell-0derived organelle matter. Resultantly, purities were even higher than those of the AEX column at 3.97 x 10^9 particles per µg of protein, and 3.12 x 10^12 particles per µg of DNA. Finally, combination of the processes showed the potential application of the chromatographic options within a larger process for exosome purification and high performance capillary electrophoresis analysis showed substantial removal of cell culture derived proteins from the recovered material, without substantial loss in particle number. The processes were assessed for potency, both individually and in sequence. No adverse effect in wound closure was noticed with all samples achieving wound closure over 90%. This showed improvement on the current gold-standard method, which could not retain product functionality