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[Display omitted] • Identifying limiting factors of reactive extraction and back-extraction with online FTIR spectroscopy. • Evaluation of reaction equilibria and reaction kinetics using FTIR spectroscopy. • Investigation on the strength of the acid-extractant complexes. • Optimized amine selection for reactive extraction and back-extraction. • Optimizing downstream concept for (di–)carboxylic acids through mechanistic understanding of complex formation. The downstream processing of biocatalytically produced (di–)carboxylic acids still is a challenging task. One innovative approach, demonstrating industrial applicability, applies an initial reactive extraction (RE) using hydrophobic amine extractants into an organic solvent, followed by a subsequent back-extraction (BE) using water soluble amine extractants into a (fresh) aqueous solution. Although showing promising results in terms of yield and selectivity, state-of-the-art amine extractant selection is only based on trial-and-error screening approaches. Thus, to enable an optimized process design and thereby optimized downstream processing, a deeper understanding of RE and BE on a molecular level is crucial. Within this work, we successfully applied an experimental setup incorporating online Fourier Transform Infrared (FTIR) spectroscopy to elucidate influencing factors on RE and BE as well as to provide a guideline for an optimized selection of amine extractants. Investigations on the kinetics of complex formation between itaconic acid (IA) and tri- n -octylamine (TNOA) in 1-decanol revealed the reaction order to be zero in TNOA, indicating that mass transfer is the rate limiting step in RE. Reaction equilibrium investigations revealed that for all IA - amine extractants combinations in 1-decanol reaction equilibrium was almost completely on the product (complex) side. The strength of the acid-extractant complexes was evaluated using FTIR experiments, providing a novel approach / methodology to efficiently select an optimal combination of amine extractants for RE and BE. Applying the novel online FTIR spectroscopy setup allowed to monitor a complete process sequence of RE and BE of IA with selected amine extractants, validating the proposed guidelines for an optimized selection of amine extractants. The results of this work will facilitate the definition of appropriate RE and BE systems for biocatalytically produced (di–)carboxylic acids. [ABSTRACT FROM AUTHOR]

[Display omitted] • Online monitoring of reactive extraction of (di-)carboxylic acids. • Simultaneous time-dependent measurements in aqueous and organic phase. • Time-dependent concentration and reaction profiles using FTIR spectroscopy. • Identification of complex stoichiometry using FTIR spectroscopy. • Optimizing reactive extraction by mechanistic understanding of complex formation. Reactive extraction (RE) using amine extractants in an organic solvent has been proven a promising downstream strategy for (di–)carboxylic acids of biocatalytic origin. Available analytical techniques allow for observation of the acid depletion in the aqueous phase and thus an indirect link to the course of the RE as well as concentration of the complex. In order to further optimize RE performance in these systems, a direct information on the complex concentration as well as its stoichiometry (amine extractant to (di–)carboxylic acid ratio in the complex) in the organic phase is crucial. Within this work, we successfully applied Fourier Transform Infrared (FTIR) spectroscopy for the detection and quantification of the acid-extractant complex formed between itaconic acid (IA) and tri-n-octylamine (TNOA) in 1-decanol. A newly developed experimental setup incorporating FTIR spectroscopy and a systematic procedure for the characterization of the acid-extractant complex allow clarifying the structure of the acid-extractant complex. Our investigations revealed the complex formation between IA and TNOA to be based on the ion-pair mechanism whereas the stoichiometry was identified to be 2:1 (TNOA:IA) when TNOA is available in excess. Applying the experimental setup enables online monitoring of the IA concentration simultaneously in both phases during extraction of IA using 1-decanol as organic extraction phase. The mechanistic understanding of the complex formation on a molecular basis gained from the results of this study as well as the possibility to online monitor RE enables the optimization of RE as downstream strategy for (di–)carboxylic acids. [ABSTRACT FROM AUTHOR]

Within the last decade, thermodynamic models have increasingly grown into the pharmaceutical industry, becoming an essential part in both, process development, as well as formulation development of small-molecule drugs. They are to an increasing degree accepted as a valuable building block to evaluate e.g. small-molecule formulation conditions. In contrast, modeling of highly complex (bio)-molecules (e.g. monoclonal antibodies) remains a challenging task and thermodynamic modeling is only rarely applied for e.g. protein formulations in the biopharmaceutical industry. Within this work, we developed a thermodynamic modeling approach to access protein-protein and protein-excipient interactions (via second osmotic virial coefficients B 22 and cross viral coefficients B 23 ) for immunoglobulin G (IgG) and bovine serum albumin (BSA) in the presence of different excipients (NaCl, sucrose, L-histidine, L-arginine) at defined buffer conditions using the e PC-SAFT equation-of-state. The pure component parameters of IgG were determined based on the amino acid sequence and further fitted to experimentally determined static light scattering signals of IgG at buffer conditions. This approach allows to predict protein-protein and protein-excipient interactions of BSA and IgG in the presence of different excipient types and thus give access to protein phase behavior in these complex solutions. Applying this approach further allows to gain a mechanistic understanding on the complex interactions in solution that govern the phase behavior, and thus can aid an optimized formulation development. • Modeling protein-protein interactions using the e PC-SAFT equation-of-state. • Novel modeling strategy for second osmotic and cross virial coefficients for proteins in the presence of excipients. • Predicting protein-protein/protein-excipient interactions in the presence of excipients based on pure component parameters. • Access to other thermodynamic quantities such as solution densities. [ABSTRACT FROM AUTHOR]

Background: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production 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 laboratory 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 processing 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. [ABSTRACT FROM AUTHOR]