Tailor-made aqueous two-phase systems for application in continuous separation of potent biomolecules

Aqueous Two-Phase Extraction (ATPE) using Aqueous Two-Phase Systems (ATPS) has long been shown to be a viable and promising alternative in the work-up of potent biomolecules (e.g. enzymes, proteins, therapeutics) from fermentation broth. Although ATPE has significant advantages over common separation strategies, such as a high biocompatibility, gentle separation profile due to low interfacial tension, good scalability and high efficiencies, industrial applications have not yet been realized. Reasons typically given are based on the ATPS “physiochemical” properties such as viscosities and low density differences between the phases, which lead to long phase separation times. However, these challenges can be addressed using advanced technology such as the “Tunable Aqueous Polymer-Phase Impregnated Resins” (TAPPIR)-Technology immobilizing one phase of an ATPS inside porous solids, which are then transferred into a chromatography column. The second aqueous phase serves as mobile phase. The main advantage of this technique is the simple and efficient emulsification and liquid–liquid phase separation through the packed-bed column design. In addition, the extraction phases, i.e. both the back extraction phase and the immobilized phase, can be reused enabling a low-waste production process. The remaining bottleneck for an industrial application is the identification of the “base” ATPS, which enables the desired extraction of the biomolecule with the required yield and purity to be competitive to existing processes. State-of-the-art ATPS design so far is based on a “trial-and-error” based approach identifying ATPS that work for a given task but often perform in suboptimal fashion. In the present work, we will present a novel thermodynamics-based strategy for the identification and characterization of tailor-made ATPS for the continuous separation of highly potent industrial enzymes by ATPE. By consideration of the molecular interactions in solution, we are able to define potentially suitable ATPS based on a predictive modeling approach using ePC-SAFT, a state-of-the-art equation of state. The objective of this step is to supply a thermodynamically optimized combination of ATPS-phase formers that lead to optimal water condition (low concentration of phase formers, large process window), in principal enabling optimal separation. This initial selection is refined by taking into account molecular interactions of the biomolecule (enzyme), by measuring and modeling biomolecule-biomolecule and biomolecule-phase former interactions. These interactions are experimentally captured using advanced light scattering techniques that are both time and cost efficient. It will be shown that, based on the description of molecular interactions through osmotic virial coefficients (B22 and B23) as well as the diffusion interaction parameter (kD) between the molecules in solution, the phase behavior of the biomolecule in an ATPS can be made accessible, but was previously inaccessible with other phase diagram estimation strategies One major advantage of our predictive modeling approach is the estimation of the partition coefficient of the biomolecule between the two aqueous phases based on a minimal set of experimental data, i.e. B22, B23, kD, and phase composition data. Furthermore, the influence of the ATPS phase-formers on protein solubility and stability can be judged qualitatively, an ideal complement in the development of ATPS. Lastly, we applied the thermodynamics-based strategy to the separation of an industrially relevant dehydrogenase from fermentation broth. The design-driven process development led to the identification of a tailor made ATPS that outperformed the reference ATPS from previous works in terms of solubility and stability of the biomolecule enabling a cost-efficient use of the TAPPIR technology

Fitting Error vs Parameter PerformanceHow to Choose Reliable PC-SAFT Pure-Component Parameters by Physics-Informed Machine Learning

State of the art thermodynamic models, such as the Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT), require a thorough parametrization (three pure-component parameters for nonassociating molecules) of the molecules considered. In our previous work (J. Habicht, C. Brandenbusch, G. Sadowski, Fluid Phase Equilibria, 2023, 565, 113657), we introduced a Machine Learning approach for a predictive parametrization of nonassociating components. Within this approach, training is performed using a Huber-loss function, comparing the ML-predicted parameter set with the original one, e.g., from literature. However, often multiple pure-component parameter sets exist for one molecule. This fact makes the training to only one “true” parameter set questionable. Within this work, we thus performed a detailed analysis on the fact of multiparameter set existence. We further expanded our ML-approach by developing a choice of two physics-informed loss functions that allow for the consideration of multiple “true” parameter sets during training. Results indicate that reliable pure-component parameters have a certain orientation when plotted in the three-dimensional parameter space. The results of this work will lead to a more reliable ML-based parametrization and ensure the prediction of optimized pure-component parameters for a given molecule

Hydroxylpropyl-β-cyclodextrin as Potential Excipient to Prevent Stress-Induced Aggregation in Liquid Protein Formulations

Due to the growing demand for patient-friendly subcutaneous dosage forms, the ability to increasing protein solubility and stability in formulations to deliver on the required high protein concentrations is crucial. A common approach to ensure protein solubility and stability in high concentration protein formulations is the addition of excipients such as sugars, amino acids, surfactants, approved by the Food and Drug Administration. In a best-case scenario, these excipients fulfil multiple demands simultaneously, such as increasing long-term stability of the formulation, reducing protein adsorption on surfaces/interfaces, and stabilizing the protein against thermal or mechanical stress. 2-Hydroxylpropyl-β-cyclodextrin (derivative of β-cyclodextrin) holds this potential, but has not yet been sufficiently investigated for use in protein formulations. Within this work, we have systematically investigated the relevant molecular interactions to identify the potential of Kleptose®HPB (2-hydroxylpropyl-β-cyclodextrin from Roquette Freres, Lestrem, France) as “multirole” excipient within liquid protein formulations. Based on our results three factors determine the influence of Kleptose®HPB on protein formulation stability: (1) concentration of Kleptose®HPB, (2) protein type and protein concentration, and (3) quality of the protein formulation. Our results not only contribute to the understanding of the relevant interactions but also enable the target-oriented use of Kleptose®HPB within formulation design

Adaptive Laboratory Evolution accelerated glutarate production by Corynebacterium glutamicum

Prell C, Busche T, Rückert C, Nolte L, Brandenbusch C, Wendisch VF. Adaptive Laboratory Evolution accelerated glutarate production by Corynebacterium glutamicum. Microbial Cell Factories. 2021;20:97.Background: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for produc-tion of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. Results: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the l-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive labora-tory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h−1 by the parental strain to 0.17 h−1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L−1 h−1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of l-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L−1, a yield of 0.23 g g−1 and a volumetric productivity of 0.35 g L−1 h−1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream pro-cessing results. Conclusion: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies

SNG und LPG aus biogenen Reststoffen - Technische Machbarkeit und Verwertungspotenzial

Dass sich biogene fett- und ölbasierte Reststoffe und Koppelprodukte durch katalytisches Cracken an Aktivkohlen und anderen porösen Katalysatoren bei Normaldruck in gasförmige Kohlenwasserstoffgemische umwandeln lassen, ist bekannt. In diesem Vorhaben werden jedoch erstmals systematische Untersuchungen dazu vorgelegt, diese Gasprodukte als Wertstoffe gezielt zu erzeugen. Erste experimentelle Ergebnisse zur Herstellung von Erdgassubstitut ("Substitute Natural Gas" - SNG) und Flüssiggas ("Liquified Petroleum Gas" - LPG) werden dabei durch eine Prozesssimulation ergänzt. Mit diesem Verfahren lassen sich nennenswerte Mengen gasförmiger n-Alkane gewinnen. Für die wichtigsten Gasbestandteile konnten Absatzpotenziale im Erdgas- und Flüssiggasmarkt, insbesondere als LPG-Beimischung zu Biomethan zur Herstellung von 100 % biobasiertem SNG für die Einspeisung ins Erdgasnetz, aufgezeigt werden. Weitere Absatzpotenziale liegen in der Verwendung im Kraftstoffbereich (Flüssiggasfahrzeuge). Dennoch wird eine wirtschaftliche Umsetzung des Verfahrenskonzeptes nur in der gleichzeitigen Wertschöpfung aus den Flüssigprodukten möglich sein. Die Verwendung zeolithischer Katalysatoren führt im Gasprodukt vornehmlich zu Ethen und Propen; bei den Flüssigprodukten sind hier teils Alkene, teils alkylierte Benzole vorherrschend. Rohstoffseitig konnten verschiedenste fettsäurehaltige Einsatzstoffe wie Fettsäurerückstände aus der chemisch-physikalischen Vorklärung eines Ölpflanzenverarbeiters, Havariefette aus der Ölpfl anzenverarbeitung, Altfett aus der Gastronomie und lebensmittelverarbeitenden Industrie oder Jatropha Curcas-Öl sowie Algenöl als Co-Feed erfolgreich eingesetzt werden. Dabei wurden an Aktivkohle bis zu 47 % energetischer Ausbeute für den organischen Anteil am Gasprodukt (OGP) erzielt. Geeignete Reaktortemperaturen für die gezielte Wertschöpfung aus der Flüssigphase lagen bei 475-500 °C. Untersuchungen zum Einfluss der Porenstruktur auf die katalytische Aktivität von Aktivkohle beim katalytischen Cracken weisen darauf hin, dass hier insbesondere die Mikroporen und kleinen Mesoporen (Poren mit 0,2-3,4 nm Radius) zum Beschleunigen der gewünschten Reaktionsverläufe notwendig sind. Gegenüber fossilen Produkten können bis zu 97 % an Treibhausgasemissionen bei der Erzeugung von 30 Gew.% Propan und 70 Gew.-% Diesel/Benzin eingespart werden