Biotechnology Progress最新文献

Perfusion culture is acknowledged as a promising platform for sustained high-density cell production, while concurrently necessitating stringent control over medium nutrient composition. A multi-component medium optimization strategy has been developed in this study, integrating the targeted feeding approach (TAFE), ¹H nuclear magnetic resonance (¹H NMR) analysis, and definitive screening design (DSD). Nine pivotal amino acids were selected through quantitative profiling of cellular uptake kinetics and literature evidence. Their concentrations were optimized using a DSD within only 24 experimental runs. The optimized formulation was demonstrated to maintain stable cell density, high viability (>97%), and excellent monoclonal antibody production in both shake flask semi-perfusion (38.63 pg/cell/day) and 3L bioreactor systems (45-61.5 pg/cell/day), while significantly reducing the accumulation of lactate and ammonium. These results suggest that the proposed strategy can effectively enhance both productivity and metabolic stability, offering excellent scalability and engineering applicability. This work provides a novel and efficient pathway for the development of perfusion culture media in biopharmaceutical manufacturing.

The metabolic dysfunction-associated steatotic liver disease (MASLD, previously formerly known as non-alcoholic fatty liver disease, NAFLD) is rapidly expanding worldwide in parrallel with the obesity pandemic. Dietary fatty acids including oleic (OA) and palmitic acids (PA) contribute to the hepatic intracellular triglyceride accumulation, and are therefore thought to play key roles in disease development and progression. Taking advantage of the cutting-edge organ-on-chip technology that mimics the 3D organ dynamic environment, we aimed at investigating the role of OA, PA and a 2:1 OA/PA mixture on the growth and function of the HepG2/C3A, a liver cell line model, over 2 and 7 days. OA supported sustained cell growth, leading to dense 3D tissues, whereas PA and OA exposure did not affect cell proliferation. PA treatment downregulated the GLUT2, INSRA, SREBP1, FASN, mRNA levels indicating a lipid metabolism perturbation in our model. The cell dysfunction caused by OA, PA, and OA/PA was associated with an increase in reactive oxygen species (ROS) production over time. Intracellular lipid monitored by oil red O was higher in cells exposed to OA than in the control ones and cells cultured with PA. Our data confirm the role of fatty acids on the growth and dysfunction of HepG2/C3A cells, and highlight distinct mechanisms through which OA and PA exert their effects.

In microbial production, regulating endogenous and exogenous synthetic pathways is essential. Techniques to induce intracellular enzyme condensation have attracted attention as a means to increase apparent enzyme activity with minimal transcriptional and translational burden on the cell. Artificial enzyme condensation can be induced by tagging enzymes with specific proteins or peptides. In our previous study, we identified novel peptide tags derived from condensate-forming Saccharomyces cerevisiae glycolytic enzymes and their potential use in controlling intracellular metabolism by inducing artificial condensates in S. cerevisiae cells. Herein, we evaluated the condensate formation of two enzymes using peptide tags and the branched violacein biosynthetic pathway from Pseudoalteromonas luteoviolacea in S. cerevisiae, and tested the effects of modulating biosynthesis to increase the production of deoxyviolacein, a byproduct with antibacterial and anticancer activities. We used several protein and peptide tags with a simplified expression system, and all the tags successfully induced artificial condensate formation in the cell. Additionally, introducing a short peptide tag successfully increased deoxyviolacein production by approximately twofold, displaying a higher efficacy compared to FUS^N, a previously reported N-terminal 213 amino acid region with an intrinsically disordered property. These results demonstrate the potential use of peptide tags to enhance bioproduction through the regulation of endogenous and exogenous synthetic pathways. The methods contribute to the development of novel strategies for microorganisms to be used for bioproduction through the controlled condensation of metabolic enzymes in cells.

High throughput process development (HTPD) has been widely adopted for efficient development and optimization of chromatographic operations in monoclonal antibody (mAb) purification. However, the integration of non-chromatographic unit operations, particularly depth filtration following protein A chromatography, which is essential for the removal of process- and product-related impurities prior to the ion exchange chromatography (IEX) operations, remains a challenge due to the absence of commercially available micro-scale depth filtration tools. This limits the integration of this unit operation within the purification sequence, restricting the analysis of process interactions and overall process understanding. In this study, a micro-scale HTPD platform was designed and evaluated to enable integration of a depth filtration mimic, Sartobind® Q anion exchange adsorber, within a mAb purification sequence. This was achieved by translating laboratory-scale protocols to the micro-scale using workflow design tools and executed on an automated liquid handling system. Step yields and impurity clearance were assessed to confirm the equivalence of scale-down. The Sartobind® Q membrane achieved effective removal of host cell DNA (hcDNA), while subsequent IEX operations removed host cell proteins (HCPs) and high molecular weight components (HMWC), meeting target product quality specifications. The platform demonstrated robustness across varying impurity profiles, supporting its applicability for diverse process intermediates. Comparative analysis with laboratory-scale operations confirmed the performance and scalability of the micro-scale system, reducing the total run time by greater than 50%. The integrated HTPD platform offers a resource-efficient, scalable approach for comprehensive mAb purification process development and is suitable for developability assessments during early-stage development.

Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell-specific and context-specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. We show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.

Key hydrodynamic-related parameters such as volumetric power input (P/V), impeller configuration, aeration strategy, and maximum gas sparge rate, as well as an appropriate feeding strategy, must be carefully selected to improve production yields in bioreactor. In this study, the feeding regimen was found to have an important impact on cell growth and productivity of a cumate-inducible CHO fed-batch cell culture. A low-volume feeding regimen avoided a rapid increase in osmolality, allowing for prolonged cell viability and a 33% increase in volumetric titer compared to the high-volume feeding regimen. Both sparged air and oxygen were used for dissolved oxygen (DO) control, utilizing three levels of airflow rates. An optimum airflow rate of 0.0031 vvm was found to improve cell growth, longevity, and thus final titer. A larger air cap required increased gas flow rates, which led to an earlier cell mortality. Scale-up from 1-L to 10-L bioreactor using constant P/V and air cap volumetric gas flow rate (vvm) allowed for comparable cell growth and productivity. Further investigation of the effect of mixing and aeration was done by maintaining P/V and vvm constant throughout the cell culture, which further improved product titers at 11 days after induction. Our study also demonstrates that keeping a constant volume by removing a culture amount equal to the feed volume added at each sampling event can significantly improve the final volumetric titer. This finding shows the benefit of developing a concentrated feed to reduce the volume increase, which in turn could greatly ease the scale-up task.

Endophytic microorganisms (EMs) residing in medicinal plants form a promising resource of anticancer compounds such as camptothecin (CPT). Given the increasing therapeutic demand for CPT, its sustainable production is of high significance. This study has investigated the EMs isolated from different parts of Ophiorrhiza mungos for the CPT biosynthetic potential. Preliminary screening of EMs for the CPT synthesis was carried out by HPLC analysis of culture extracts, and the HPLC-positive extracts were further confirmed via LC-MS/MS. From a total of 175 EMs screened in the study, 17 strains (14 bacterial and 3 fungal) were found to be CPT producing, with most of them being sourced from the root tissues. Among the bacterial strains, Alcaligenes faecalis subsp. phenolicus S18 exhibited the highest CPT yield (1294.52 μg/L) followed by Bacillus tequilensis (309.02 μg/L). From the fungal strains, Aspergillus sp., S109, S42, and S111 yielded CPT of 22.07, 18.98, and 13.26 μg/L, respectively. Overall, CPT yield among the bacterial producers ranged from 1294.52 to 5.16 μg/L, predominantly from the Bacillus, Acinetobacter, Alcaligenes, and Pseudomonas genera. This study provides the first report on the CPT production by A. faecalis and Aspergillus sp. isolated from O. mungos, and also the first documentation of CPT synthesis in Stenotrophomonas, Fictibacillus, Acinetobacter, and Pseudomonas genera. These findings highlight the potential of novel microbial sources as high-yielding, reliable, and cost-effective alternatives to support commercial CPT production.

Achieving consistent CHO cell culture performance during process scale-up is critical but often challenged by subtle changes in operational parameters. This study investigates how differences in feed media filtration and storage during scale-up can impact CHO cell culture performance. A 70% reduction in titer and a 25% drop in peak viable cell density (VCD) were observed at 2000 L scale. Root cause analysis revealed that the secondary filtration of feed media was likely a contributing factor. Trace element analysis confirmed significant copper(II) ions (Cu²⁺) loss in feed media at 2000 L, likely due to precipitation during storage and subsequent removal by secondary sterile filtration. This resulted in continued lactate accumulation and reduced titer. Feed storage conditions had an impact on Cu²⁺ stability, with room temperature storage accelerating Cu²⁺ loss when compared to storage at 2 to 8°C. By eliminating the secondary filtration step and optimizing feed media storage conditions, process performance was successfully restored at 2000 L scale, matching smaller scale performance. This study highlights how feed filtration and storage critically affect micronutrient stability and availability during scale-up. While secondary filtration may be used for additional microbial control, it can inadvertently alter feed composition, affecting cell metabolism and productivity. Thorough evaluation of feed stability, filtration, and storage strategies is therefore key to ensuring consistent bioreactor performance across scales.

Probiotic use has become more important in aquaculture for healthy and sustainable output. In particular, Bacillus spp. have emerged as effective probiotic agents, improving gut health, enhancing the immune system, promoting growth, and providing protection against pathogens in fish. Therefore, the application of Bacillus in aquaculture offers a strategic approach to increasing productivity while reducing the reliance on antibiotics. In this study, the antibacterial activities of Bacillus isolates, whose probiotic properties will be determined, against test bacteria that are fish pathogens such as Aeromonas hydrophila, Vibrio anguillarum, Lactococcus garvieae, and Yersinia ruckeri were determined by using cross-streak method and agar well diffusion methods. Then, antibiotic resistances of 75 isolates determined to have antibacterial activity were screened against 9 different antibiotics by the agar disc diffusion method. Gastric juice (pH 2.5) tolerance of 55 isolates determined to be sensitive to antibiotics was examined, and the tolerance of 13 isolates to gastric juice was determined. Optimum growth characteristics at acidic pH, surface hydrophobicity, bile tolerance, and protease, amylase, lipase, and cellulase activities, hemolytic activities, coagulase activities, bacterial adhesion abilities, and biofilm production properties of these isolates were determined. As a result, Bacillus subtilis Ö-4-68, with the best probiotic properties, was selected from the examined isolates, and production medium optimization was carried out with laboratory scale statistical experiment design (Response Surface Methodology, RSM) for high amount of biomass production. As a result of the trials, an economical cost-effective production medium content with high biomass production was determined.

Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC-encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto-oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled-up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol-chloroform precipitation and multiple biophysical analyses (SDS-PAGE, SEC-HPLC, MALDI-TOF, and LC-MS/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.