Biotechnology Journal最新文献

Variation in the primary sequence of monoclonal antibodies (mAbs) can negatively affect their behavior in biopharmaceutical manufacturing platforms, and efforts to identify mAbs with poor “developability” characteristics lack robust methods for assessing mAb expression from an industrially relevant platform. Recent advancements in site-specific integration-based (SSI) platforms in Chinese hamster ovary (CHO) cells can mitigate the high transcriptional variation observed with random integration and the low industrial relevance of transient expression by providing a flexible platform for mAb expression from a consistent clonal background. This work applies a novel SSI-based expression system capable of generating isogenic cell pools in less than 1 month to systematically compare the expression of ten sequence variants of two therapeutically relevant mAbs from two genomic loci under industrially relevant culture conditions. Eight single amino acid mutations in trastuzumab resulted in reduced productivity compared to the wild-type mAb in batch cultures, and three mutations maintained a low-expressing phenotype in fed-batch cultures. The mutations resulted in variant-specific patterns of decreased domain stability and increased ER stress. The application of industrially relevant SSI systems in developability workflows could strengthen the understanding of the sequence determinants of mAb expression to improve mAb design, candidate selection, and process development decisions.

3D printing is emerging as a promising fabrication technique for microfluidic devices. In this work, this technology was exploited in the development of a microfluidic chromatographic column with nominal volume of 54 µL. The microcolumn was packed with a cation exchange resin and characterized, using potassium iodide as a tracer, in terms of porosity (ε = 0.72), plate number, and asymmetry factor (0.8 < A_S < 1.8 for flowrates >50 µL/min). To showcase the potential of this microdevice, it was exploited in the characterization of the chromatographic behavior of lysozyme. The measured saturation capacity (q^∞= 88.14 g/L_resin at 340 cm/h) was in line with the manufacturer declaration (85–135 g/L at <500 cm/h). In addition, the effect of NaCl at different concentrations on the protein adsorption isotherm was characterized, demonstrating a Langmuir to anti-Langmuir transition at concentrations ≥300 mM. The axial dispersion coefficient was finally determined ($�D_{�A�X�}$= 6.7 · 10⁻⁹ m²/s). In this way, the mcirofluidic column allowed to develop a comprehensive mechanistic model describing the transport of lysozyme in the chromatographic medium using only 30 µL of resin and <1 g of protein, addressing the issue of limited availability of biomolecules and streamlining the process development.

Recent advancements in stem cell research forge them into one of the most promising sources for cell therapy applications. Quality monitoring in stem cell culture is essential for ensuring consistency, viability, and therapeutic efficacy. Traditional methods involve periodic sampling for conducting endpoint assays such as cell viability, proliferation, and differentiation using microscopy and flow cytometry, which are labor-intensive and often lack the real-time monitoring of the processes for scale-up applications. This paper explores artificial intelligence (AI)-driven approaches for real-time quality control, integrating machine vision, predictive modeling, and sensor-based monitoring. AI models analyze high-resolution imaging and multi-sensor data to dynamically track critical quality attributes (CQAs), including cell morphology, proliferation rate, differentiation potential, environmental stability (pH, oxygen, and nutrient levels), genetic integrity, and contamination risks. These models enable automated anomaly detection, differentiation tracking, and adaptive culture optimization. By leveraging real-time feedback systems and multi-omics integration, AI-driven techniques enhance scalability, reproducibility, and process automation in stem cell biomanufacturing. This review outlines current advancements, challenges, and future directions in AI-assisted quality monitoring and highlights its potential to improve fully automated, scalable production of stem cell lines for clinical translation and regulatory compliance in regenerative medicine.

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in gene therapy, yet the molecular interactions underlying rAAV production in host cells remain incompletely understood. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs), are increasingly recognized as key regulators of viral and cellular processes. This study investigates the dynamic expression profiles of miRNAs and snoRNAs during rAAV plasmid transfection and vector production in HEK293F cells. A total of 142 miRNAs were differentially expressed during the peak phase of rAAV production, with 128 associated with the Gene Ontology term “viral process”, indicating broad involvement in host-virus interactions. Target gene analysis linked these miRNAs to biological pathways such as nucleocytoplasmic transport, innate immunity, apoptosis, and transcriptional regulation, highlighting potential roles of miRNAs in shaping the cellular environment during viral vector assembly. In contrast, snoRNAs exhibited more modest changes in expression, yet five were significantly differentially expressed during active production, suggesting a possible, underexplored involvement in viral replication. These findings illuminate the underexplored contributions of ncRNAs to the host response during rAAV biogenesis and provide a valuable resource for understanding how cellular regulatory networks are engaged throughout vector production.

Microbial reduction of toxic Se(IV) oxyanions to biogenic Se(0) has garnered considerable attention for detoxification. This study presents a comprehensive investigation of Se(IV) reduction by environmentally versatile Exiguobacterium genus through integrated physicochemical, genomic, and transcriptomic analyses. Exiguobacterium mexicanum PY14 demonstrated remarkable efficiency, reducing ∼1 mM selenite to extracellular Se(0) within 12 h under aerobic conditions, with broad adaptability to pH (7–9), temperature (30–37°C), and salinity (up to 40 g L⁻¹ NaCl). The produced Se(0) revealed crystalline nanoaggregates with biomolecular coatings. Genomic sequencing identified a chromosome and six plasmids enriched with genes for carbohydrate metabolism, inorganic ion transport, and mobile genetic elements. Transcriptomic profiling under Se(IV) stress unveiled a coordinated stress response: up-regulation of catabolic pathways (glycolysis and citric acid cycle) for energy and NAD(P)H production, bacterial motility, and chemotaxis, alongside down-regulation of energy-intensive biosynthetic processes. Notably, genes for glutathione biosynthesis (gsh), NAD(P)H generation (gntZ), and ROS scavenging (btuE) were significantly up-regulated, along with the evidence of increased GSH levels, implicating a GSH-dependent detoxification pathway driving Se(IV) reduction. These findings deepen mechanistic understanding of Se(IV) reduction mechanism within the understudied Exiguobacterium genus, and the strain's haloalkaliphilic trait underscores its potential for bioremediating Se(IV)-contaminated saline-alkaline environments.