This work presents a methodology for the generation of continuous fibre trajectories based on principal stress directions in continuous fibre-reinforced additive manufacturing (CFAM). The material system considered consists of continuous carbon fibre (CCF-1.5K) embedded in a CFC-PA thermoplastic matrix. CFAM enables the deposition of fibres along tailored paths, allowing improved alignment with the load direction, compared to traditional composite manufacturing. In this way, the strong anisotropy of composite materials, typically considered a limitation, is exploited as a design opportunity by aligning fibres with the structural load paths. The proposed approach combines finite element analysis with a path generation procedure, including the computation of principal stress directions, the extraction of streamlines of the principal stress field, and a dedicated post-processing stage aimed at obtaining continuous and manufacturable fibre layouts. The effectiveness of the method is assessed through a finite element-based comparison with conventional fibre configurations, showing an increase in global stiffness of approximately 20% with respect to the best-performing unidirectional layout. In addition, the feasibility of the generated trajectories is demonstrated through printing tests performed on a continuous fibre additive manufacturing system. The results confirm that the proposed methodology enables the generation of physically realizable fibre paths while improving structural performance.
Process analytical technology (PAT) is a framework that encourages the implementation of analytical tools for real-time monitoring of critical batch components during the production of pharmaceuticals. In mammalian cell cultures, Raman spectroscopy as a PAT tool has demonstrated its value for accurate in-line monitoring of various process parameters as well as quality attributes. However, as process development cultures are transferred to manufacturing scales, the small-scale models are often adjusted either with new calibration data or with transfer learning algorithms. This model adjustment step makes the overall process transfer to manufacturing less straightforward from an analytical standpoint, requiring additional resources and specific data treatment expertise to fine tune the models. In this work, we compare a specific spectra processing approach against existing approaches reported in the literature for processing Raman spectra in the context of scale-up challenges. The proposed novelty is a preprocessing strategy which computes spectra normalization based on the 3100-3700 cm-1 band. Data generated at 3 L scale were used to monitor a 3000 L bioreactor without model recalibration or transfer learning with equivalent accuracy as a same scale 3 L bioreactor monitoring. While broader validation case would be warranted, these results demonstrate a direct scale-up model implementation in large scale manufacturing under the studied conditions.
Venipuncture is a fundamental clinical skill that requires repeated practice in a safe learning environment before being performed on patients. However, commercial venipuncture simulators are often expensive and not always available in sufficient numbers for repeated student training. This study aimed to design, manufacture, and evaluate a low-cost, reusable venipuncture training model for medical education. The simulator was developed using a silicone-based soft-tissue model with embedded silicone tubing simulating superficial veins at different depths. A passive hydrostatic pressure system was used to provide visual feedback during successful venipuncture. The model was manufactured using a 3D-printed mold, platinum-cure silicone, silicone tubing, and a porous support layer to improve durability and fluid absorption. The total material cost per model was approximately 4-5 EUR. The model was used in a practical training session with 50 medical students as part of a scheduled educational activity. Following the training, participants were invited to complete a voluntary and anonymous questionnaire designed to evaluate the perceived realism, usability, and educational value of the model. The questionnaire consisted of 10 items rated on a 5-point Likert scale. The instrument was developed specifically for this study and was not previously validated; therefore, the results should be interpreted as exploratory measures of user perception. Students reported high satisfaction across all evaluated categories, particularly ease of use, the ability to perform repeated venipuncture, and the visual feedback system. This low-cost, in-house manufactured venipuncture simulator provides a practical and sustainable solution for procedural training. Its affordability, reusability, and ease of manufacturing make it suitable for widespread use in medical education, particularly in settings with limited access to commercial simulation equipment.
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Balancing the strength and toughness of materials remains a long-standing core challenge in materials science. In this study, a novel design strategy for Al2O3/Mg interpenetrating phase composites (IPCs) is proposed. Al2O3 ceramic scaffolds with two types of Split-P triply periodic minimal surface (TPMS) structures (shell-type and solid-type) were fabricated via stereolithography (SLA) and then infiltrated with AZ91D magnesium alloy using lost foam casting (LFC), and consequently, Al2O3/Mg IPCs were successfully fabricated. In the proposed method, cocontinuous interpenetration of ceramic and metal phases is achieved, ultimately forming a dense interlocking structure with no interfacial delamination and a relative density exceeding 95%. Quasi-static compression tests and finite element simulations confirm that the as-built composites exhibit a compressive strength of up to 187.5 MPa and a specific energy absorption of 11.18 J/g; among them, the solid-type IPC shows a slightly higher compressive strength, while the shell-type IPC demonstrates superior energy absorption performance. The excellent performance of the IPCs is attributed to the TPMS structure that effectively mitigates the local stress concentration prone to occur in traditional truss structures, along with the synergistic strengthening effect between ceramic and metal phases that substantially improves the plastic deformation capacity and energy dissipation efficiency of the composites. This study provides a new idea for the design and fabrication of high-strength and lightweight composites, which hold significant application potential in lightweight load-bearing and impact-resistant energy-absorbing fields.
Human induced pluripotent stem cells (iPSCs) are a promising starting material that can be differentiated into therapeutic cell products for clinical trials. The production of clinically applicable drug products requires good manufacturing practice (GMP)-compliant iPSCs as the initial starting material. Under the framework of the Korean Ministry of Food and Drug Safety, we generated a master cell bank (MCB) of a human iPSC line, KNIH01-MCB, from peripheral blood mononuclear cells obtained from a healthy female donor under a GMP system. To verify cell identity and quality, we performed a risk assessment of genetic stability, pluripotency, cell line identity, and sterility. For this purpose, we used validated methods, including polymerase chain reaction, karyotyping, short tandem repeat analysis, ABO/human leukocyte antigen (HLA) typing, and bacterial growth testing. After material qualification, the cells underwent comprehensive quality testing, including viral screening, ABO/HLA typing, and sterility testing. We successfully established an MCB of the iPSC line, enabling clinical readiness, including full traceability and donor eligibility for clinical applications. The MCB met the quality control criteria of our in-house GMP system. These iPSCs provide a GMP-compliant and clinically applicable starting material for therapeutic cell manufacturing intended for human use. Induced pluripotent stem cells (iPSCs) are cells that can develop into almost any cell type in the body, making them a valuable tool for developing new medical treatments. In this study, we generated good manufacturing practice-compliant iPSCs from the blood cells of a healthy female donor using a non-integrating episomal vector, which avoids permanent changes to the cells’ DNA. We performed extensive testing to confirm that the reprogramming vector was fully cleared and that the cells remained genetically stable and free of contamination. We also verified their identity and quality using multiple laboratory methods. These results support the use of our iPSCs as a reliable and safe starting material for future clinical applications. These cells can be used to produce various differentiated cells, including hematopoietic cells such as red blood cells and platelets, for clinical use.
Biopharmaceutical manufacturing has been using ultrafiltration (UF) and diafiltration (DF) for buffer exchange, desalting, and formulation of biologics. The legacy UF/DF is commonly a two-step batch process that is challenging to integrate into end-to-end continuous biomanufacturing. Here, we introduce asymmetric dialysis, a novel one-step continuous process that combines UF and DF. It works by utilizing asymmetric flow between the inlet and outlet of the retentate and complementary flow of the dialysate solution, achieving product concentration, buffer exchange, and salt removal using a commercially available hollow fiber device. Asymmetric dialysis can achieve product concentrations of 105 (3.8×), 200 (10×), and 64 g/L (9.4×) starting from feed concentrations of 30, 20, and 7 g/L, respectively, with modest pressures of 6-7 psi. The interplay between feed and exchange buffer flow rates was exploited to make the process sustainable by reducing buffer consumption by 75% (25 L/kg mAb) compared to conventional batch UF/DF (100 L/kg, mAb). We successfully processed 7 kg of mAb at 20 g/L feed using 5-day asymmetric dialysis with a daily productivity of 0.8 kg/m2/day to product concentration of 200 g/L. These results demonstrate the potential of asymmetric dialysis, a simple, sustainable, and low-cost bioprocessing technology for continuous bioprocessing.
This investigation uses polycrystalline cubic boron nitride (PCBN) tools for precision turning of D6AC (45CrNiMoVA) hardened steel, thereby enabling the manufacturing of components that meet the requirements of intelligent manufacturing lines. A Taguchi's L16 (43) orthogonal design was employed to systematically investigate the effects of cutting speed, depth of cut, and feed rate on cutting force, cutting temperature, surface roughness, and tool wear. Analysis of variance (ANOVA) was then conducted to quantify the contribution of each cutting parameter, and high-accuracy predictive models (R2 > 0.86) were established for the key response variables, namely cutting force components (Fx, Fy, Fz), cutting temperature (T), and flank wear width (VBmax). The results show that excellent surface quality can be achieved within the investigated range, namely at cutting speeds of 100-250 m·min-1, depths of cut of 0.05-0.2 mm, and feed rates of 0.05-0.125 mm·rev-1, with surface roughness (Ra) below 0.8 μm and mostly around 0.4 µm. At a feed rate of 0.05 mm·rev-1, the measured Ra was greater than the theoretical value (Ra*), whereas at a feed rate of 0.075 mm·rev-1, Ra was lower than Ra*, with the difference increasing as feed rate increased. The ANOVA results showed that cutting forces were dominated by depth of cut, cutting temperatures by feed rate, and tool wear by depth of cut. The optimal process strategy was derived as follows: first, prioritize a lower feed rate; second, select an appropriate depth of cut based on tool failure or deformation control objectives; and third, choose a suitable cutting speed according to tool-life requirements or machining efficiency. This study provides process guidance and predictive tools for PCBN finishing of D6AC steel, thus promoting green, precise, and efficient machining of high-strength, high-hardness, and low-thermal-conductivity materials.
Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.
The food enzyme endo-1,4-β-xylanase (4-β-d-xylan xylanohydrolase; EC 3.2.1.8) is produced with the genetically modified Bacillus licheniformis strain NZYM-FX by Novozymes A/S. The production strain meets the requirements for the qualified presumption of safety (QPS) approach. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in two food manufacturing processes. Since residual amounts of food enzyme-total organic solids are removed in both processes, dietary exposure was not calculated. Given the QPS status of the production strain, in the absence of concerns resulting from the food enzyme manufacturing process and taking into account the negligible dietary exposure under the intended uses, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the endo-1,4-β-xylanase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.
The food enzyme with aspergillopepsin I (EC 3.4.23.18) and carboxypeptidase C (EC 3.4.16.5) activities is produced with the non-genetically modified Aspergillus sp. strain ACP 112-311 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in 14 food manufacturing processes. Since residual amounts of food enzyme-Total Organic Solids (TOS) are removed in one process, dietary exposure was calculated for the remaining 13 food manufacturing processes. It was estimated to be up to 0.079 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 180 mg TOS/kg bw per day, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 2278. A search for the homology of the two amino acid sequences to known allergens was made and matches with one food, three respiratory and one injected allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.
Efficient and scalable isolation of specific cell populations remains a central bottleneck for genome engineering, pooled screening, and cell therapy manufacturing. Here, we present DASIT ( D estabilized-nanobody A ntigen S election and I dentification T ool), a protein-based circuit for antigen-specific cell selection. DASIT uses a destabilized nanobody fused to an antibiotic resistance protein. In cells expressing the target antigen, binding of the nanobody fusion to the cognate antigen stabilizes DASIT, thereby coupling the presence of an antigen to a selectable signal. We developed DASIT circuits that enable robust selection of antigen-expressing cells and show that they can be designed to target distinct antigen classes and perform across cell types. Because DASIT operates at the protein level, it supports both stable integration and transient delivery, enabling recyclable selection without permanent genomic integration of resistance markers. We demonstrate scalable, FACS-free enrichment in three challenging applications: multiplexed, logic-gated integration of landing pads in human iPSCs, high-throughput CRISPR screening, and phenotypic selection of in vitro -derived neurons at transplantation scale. By decoupling selection from vector integration, DASIT establishes an automation-compatible architecture for multistep genome engineering, high-throughput library screening, and large-scale cell manufacturing. DASIT enriches for antigen-positive cells across multiple selection markers and antigensIntermediate levels of DASIT expression support selection across stable and transient delivery modalitiesLogic-gated, precision genome engineering of human iPSCs via DASIT selectionDASIT enables scalable activity-based selection for high-throughput base editing screensDASIT-selected engineered motor neurons survive grafting into acute spinal cord injury.
Chinese hamster ovary (CHO) cells serve as the backbone of modern large-scale manufacturing of monoclonal antibodies. Central to this process are fed-batch cultures, where cells are grown from low to high cell densities and go through a mAb production phase. Despite CHO cells' widespread usage and vital role in the production of biologics, the cellular states during fed-batch coinciding with high specific productivity and apoptosis are poorly understood. Crucial to this understanding is a clear depiction of the various subpopulations of cells that exist in a fed-batch culture over time. In this work, an Ovizio iLine F PRO was used to image the cells in benchtop bioreactors and gather morphological and optical information for several CHO cell lines. A cluster analysis was applied to the Ovizio iLine F PRO raw data, revealing a diverse set of cellular sub-populations within each culture. Raw data from the Ovizio were then transformed into tank-level data and applied to offline measurements for viability and apoptosis in regression analysis. We used the results from the cluster analysis and feature averaging to set up regressions for offline measurements. Our regression analysis illustrates the power of in-line imaging of CHO cells as a rich process analytical tool.
Regulatory reliance pathways are increasingly being recognized as essential tools to accelerate patient access to medicines globally while building National Regulatory Authorities (NRA) capacity and capability. Although significant progress has been made in a number of international pilots for Chemistry, Manufacturing and Controls (CMC) post approval changes, there has been more limited application to labelling variations including indication extensions. This paper describes the first reliance pilot for an indication extension, which engaged 21 NRAs across multiple regions. The pilot leveraged the European Medicines Agency (EMA) as a reference agency and incorporated a digital platform, whose use was optional by the NRAs involved, for real-time sharing of questions and responses. The objectives were to reduce approval timelines, promote regulatory convergence, streamline health authority questions and enhance transparency among participating authorities. Results demonstrated significant reduction in approval timelines, with an average reduction of 3 months per country compared to standard timelines across participating countries. This article outlines the process of establishing the pilot, including planning and engagement with regulatory authorities, analysing success factors and challenges encountered including recommendations for optimising the process further. The findings suggest that regulatory reliance for indication extensions can substantially improve efficiency in regulatory processes, reducing approval timelines and ultimately benefiting a wider patient population to access medicines globally and timely.
Aromatic hydrocarbons such as benzene, toluene, and ethylbenzene are extensively used as solvents in coatings, resin, and artificial leather industries. Azeotropic mixtures involving these compounds are commonly encountered in chemical manufacturing, where accurate azeotropic temperature and composition are essential for designing and optimizing separation processes such as extractive and pressure-swing distillation. In this study, two quantitative structure-property relationship (QSPR) models were developed to predict the azeotropic temperature and composition of binary mixtures containing aromatic hydrocarbons using only molecular structural information. The models show excellent agreement with experimental data (R2 = 0.9454 and 0.9448, R adj 2 = 0.9400 and 0.9413). Internal validation via leave-one-out cross-validation yields R cv 2 = 0.9308 and 0.9364, while external validation using an independent test set yields Q ext 2 = 0.8939 and 0.9364, indicating strong robustness and superior predictive performance compared to previously reported models. Molecular geometries were optimized using HyperChem 8.0, employing MM + and PM3 methods. Molecular descriptors were calculated using the Online Chemical Modeling Environment (OCHEM). Binary mixture descriptors were derived from pure-component descriptors via Kay's mixing rule. The genetic function approximation (GFA) algorithm was used to select the most relevant descriptors, and predictive models were constructed using multiple linear regression (MLR). Model robustness and predictive capacity were evaluated using leave-one-out cross-validation and an external test set, with applicability domains assessed via Williams plots. All computational procedures and modeling analyses were performed using OCHEM, SPSS, and HyperChem 8.0.
The escalating burden of musculoskeletal disorders, such as osteoarthritis, osteoporosis, inflammatory arthritis, bone tumors, and skeletal infections, necessitates precisely targeted therapeutics beyond conventional interventions. Nucleic acid aptamers are a next-generation ligand class distinguished by their high affinity, target specificity, low immunogenicity, and programable chemical properties. Their incorporation into nanoparticles, DNA nanostructures, hydrogels, microneedles, exosomes, and implant coatings is reshaping the therapeutic landscape of bone and joint diseases. This review synthesizes preclinical evidence from in vivo and ex vivo models, highlighting aptamer-functionalized carriers for targeting bone resorption, cartilage damage, synovial inflammation, bacterial infections, and skeletal malignancies. A classification framework based on aptamer target types, including cellular, extracellular matrix, signaling pathway, and pathogen-specific ligands, is mapped to appropriate nanocarriers and delivery routes. Key engineering parameters, including dissociation constant, ligand density, multivalency, linker design, particle size, and surface charge, are critical determinants of biodistribution, tissue penetration, and target specificity. Bone-to-reticuloendothelial system ratio and joint tissue retention metrics were proposed to guide rational design. Safety profiles, immunogenicity, and manufacturing feasibility were integrated into a translational roadmap. Standardized reporting protocols and priority indications, including intraarticular delivery in arthritis, bone defect regeneration, and targeted delivery in bone malignancies, have been identified to facilitate clinical translation.
Wire arc additive manufacturing (WAAM) demands aluminum feedstock with tightly controlled diameter and high surface integrity. Adding hard TiC nanoparticles is a viable route to enhance the mechanical response of Al wires, yet the associated increase in contact severity can accelerate the wear of wire processing tools, particularly cemented carbide dies. This study elucidates the unidirectional sliding interaction between a TiC reinforced Al WAAM wire, and a WC/Co die material containing 5 wt% Co, using a modified scratch testing configuration under dry and lubricated conditions. Two dominant mechanisms are identified: (i) aluminum adhesion on the die surface and (ii) third body abrasion arising from WC particle pull out, promoted by preferential degradation of the cobalt binder. The presence of TiC nanoparticles reduces both the extent of Al transfer and the intensity of third body abrasion, an effect that is further amplified by lubrication. Consistently, lubrication also diminishes surface defects on the wire after sliding. The results provide a mechanistic basis to balance wire strengthening with tool life and highlight practical levers-nanoparticle reinforcement and lubrication strategies-for mitigating die damage while preserving WAAM wire surface quality.
Recent clinical trials have highlighted the potential of P63+ lung progenitor cell (LPC) transplantation for lung repair and regeneration. Currently, ex vivo P63+ LPC expansion depends on coculture with growth-arrested fibroblast feeder cells (GAFs), necessitating repeated purification during passaging. While differential enzymatic digestion (DED) and fluorescence- or magnetic-activated cell sorting techniques (FACS/MACS) offer partial solutions, a scalable, efficient, and consistent separation technique remains unmet, particularly for cell therapy manufacturing. Here, we present a multidimensional double spiral (MDDS) inertial microfluidic device designed for high-throughput label-free enrichment of P63+ LPCs. The MDDS device achieves cell size-based separation of P63+ LPCs and growth-arrested feeder cells, processing at a rate of 106 to 107 cells per minute. MDDS-sorted P63+ LPC purity correlates with the LPC-to-GAF ratio in culture. With an initial ratio >1:1, it yields P63+ LPC purity exceeding 80%. Moreover, the device consistently recovers >80% of P63+ LPCs, with the unrecovered fraction enriched in senescent cells exhibiting compromised clonogenicity and differentiation capacity. In direct benchmarking against DED and FACS, the MDDS device delivered a balanced performance in terms of purity and recovery, while offering advantages in throughput, consistency, and scalability. We propose that this technology could enable more consistent and efficient enrichment of feeder-cultured P63+ LPCs, thereby supporting more robust clinical manufacturing processes.
Truss-like and minimal surface-based cells are among the promising candidates for novel impact-resistant structural designs. However, the influence of cell configurations on impact resistance performance remains unclear. In this paper, the energy absorption characteristics of three truss-like cells (BCC, Fluorite, and Diamond) and three minimal surface cells (Gyroid, Primitive, Diamond) are systematically compared using quasi-static compression experiments and refined numerical models. Experimental results indicate that minimal surface cells possess clearly superior specific energy absorption performance. Specifically, the Gyroid (G-surface) exhibits a specific energy absorption (25 kJ/kg) approximately 2.3 times greater than the highest value among truss-like cells (11 kJ/kg), accompanied by an extended plateau strain by about 20%. Additionally, due to stress concentration at joints, truss-like cells show notably lower plateau forces compared to minimal surface cells. However, truss-like cells demonstrate better manufacturing precision and quality control, as evidenced by a relatively small average weight deviation (about 1.2%). Furthermore, numerical simulations were conducted to explore differences in deformation mechanisms between two representative cells. Results reveal that the BCC structure absorbs energy through localized shear band formation induced by point plastic hinges, whereas the Primitive (P-surface) minimal surface structure achieves more uniform plastic deformation via distributed line plastic hinges. Finally, impact simulations of protective structures show that the maximum stress in the P-surface-filled structure is reduced by 4.6% compared to the BCC-filled structure, and stress distribution uniformity is improved by 37%. The findings from this study provide valuable references and data support for future anti-impact structural designs.
The electrocatalytic chlorine evolution reaction (CER) is essential to modern chlor-alkali industry, yet conventional RuO2 catalysts suffer from parasitic oxygen evolution. High-entropy ruthenium oxides (Ru-HEO) are promising alternatives, but their practical design is hindered by complex composition-structure-performance relationship. Herein, we construct a Pareto-guided multi-objective Bayesian optimization framework to enable autonomous high-throughput exploration of quinary Ru-HEO system. Through this trade-off strategy, we identify compositions that efficiently balance mass activity, Cl2 selectivity and material cost. The leading Ru-HEO catalyst with only 8.4 at% Ru achieves a remarkable activity of 5083 A g-1 Ru at 1.50 V versus RHE and maintains excellent 100-h stability, outperforming commercial RuO2 and the state-of-the-art catalysts reported. Integrated into a photovoltaic-electrochemical (PV-EC) prototype device and tested under simulated diurnal illumination, it sustains >95% selectivity, a maximum solar-to-chemical (STC) efficiency of 14.6% and projected Cl2 production costs as low as $0.177 per kg. Our work establishes a closed-loop, AI-accelerated research paradigm that integrates multi-objective optimization with robotic experimentation, offering a generalizable and expedited pathway toward high-performance electrocatalysts for sustainable chemicals manufacturing.