Proteinogenic amino acids are important supplements in foods, feeds, and pharmaceuticals. However, the development of cost-efficient and scalable platform downstream processes for multi-ton manufacturing of amino acids is complicated by upstream process heterogeneity resulting in plethora of implementations at laboratory and industrial scale. Here, we review 18 unit operations and analyzed >50 downstream processes reported over six decades to compare separation performance, sustainability and costs, highlighting process advantages and limitations. We find that chromatography is used in >65% of downstream processes, achieving purities >98% in different setups. Sustainable and cost-effective alternatives seem to challenge this position. For example, membrane separations can achieve purities of 60-98% with reduced environmental footprint, costs, and additional options for process integration, mainly for acidic and basic amino acids. Crystallization is frequently used for formulation and purification if >50 g L-1 of amino acids with extreme pI are processed. Options to streamline current processes are discussed.
Bone marrow stromal cells (BMSCs) are classified as Advanced Therapy Medicinal Products (ATMPs) in the subcategory of Cell Therapy Medicinal Products (CTMPs), and the production of these cells for clinical use under good manufacturing practice (GMP) standards is regulated by the European Medicines Agency (EMA). To meet GMP standards for CTMPs, the accuracy and precision of cell-counting methods are essential for monitoring in-process quality and controlling clinical dosing. To achieve this purpose, we validated an automated cell-counting method for evaluating cell concentration and viability, enabling large-scale analysis and reducing analyst-associated variability. Automated cell counting was performed using the LUNA™ automated cell counter, and we validated the parameters against the hemocytometer using the ICH Q2R1 framework. The results showed that automated cell counting yielded acceptable accuracy (90–110%) and RSD (<10%). This demonstrates that LUNA™ automated cell counters may provide an efficient alternative for measuring BMSC cell concentration and viability.
Historically, achieving efficient filtration of fibrinogen solutions using virus removal filters has been challenging due to the fibrinogen’s tendency to aggregate and its molecular size and structure. In this study, the feasibility of fibrinogen using the filtration the Planova™ 20 N filter and its virus removal efficacy under optimized filtration conditions were investigated. Optimization of arginine (400 mM) and fibrinogen (2 mg/mL) concentration in the solution, along with pre-filtration to remove the aggregates, was essential for achieving efficient filtration. Notably, Planova 20 N filter exhibited robust virus removal performance and the biological activity of fibrinogen including its clotting function was preserved, indicating that protein integrity was maintained throughout the process.
Lactic acid bacteria (LAB), such as Limosilactobacillus fermentum are persistent contaminants in bioethanol fermentation facilities and inhibit Saccharomyces cerevisiae, reducing ethanol yields. Rising resistance to antibiotics such as virginiamycin makes contamination control increasingly difficult. Recombinant endolysins such as LysKB317 provide a promising alternative. To improve LysKB317 performance, five variants were engineered by duplicating and rearranging its enzymatically active (EAD) and cell binding (CBD) domains. These constructs were compared to the wild-type endolysin. Differential scanning calorimetry (DSC) and bacterial lytic assays demonstrated that domain shuffling altered bacteriolytic activity and thermostability. The wild-type LysKB317 exhibited the highest initial lytic activity (1.16 × 10-4 OD/s‧µM), whereas LysKB317EADx3-CBD displayed comparable bacteriolytic kinetics (7.99 × 10-5 OD/s‧µM) and LysKB317CBD-EAD-CBD (ΔCp = 24.66 kJ/mol‧K) exhibited the greatest thermostability. Despite reduced activity, both variants effectively control LAB contaminants and prevented stalled corn mash fermentation. Domain shuffling demonstrated effective bacterial control strategy for generating novel endolysins for biocontrol.
Cadmium (Cd), a toxic heavy metal, poses a significant threat to aquatic ecosystems by impairing primary producers such as microalgae. This study investigated the adaptive responses of Chlamydomonas reinhardtii to Cd stress through physiological, biochemical, and GC-MS-based lipidomic analysis. Cultures were exposed to the EC50 concentration of Cd (20 ppm) for 8 days. Cd exposure significantly increased oxidative stress markers, including hydrogen peroxide (+69%), malondialdehyde (+336%), and proline (+144%). Total carbohydrate and lipid contents increased by 21.7% and 14%, respectively, whereas protein and total photosynthetic pigment contents decreased by 39.5% and 44%, respectively, indicating metabolic reprogramming under Cd stress. Antioxidant capacity was enhanced as reflected by a 50% reduction in the DPPH IC50 value. Lipidomic analysis revealed a decrease in unsaturated fatty acids accompanied by the accumulation of lipid-derived aldehydes and saturated hydrocarbons. KEGG pathway enrichment analysis identified significant alterations in fatty acid biosynthesis, fatty acid elongation, glutathione metabolism, and butanoate metabolism, highlighting coordinated oxidative and lipid metabolic reprogramming as key adaptive responses to Cd stress.
