Biology subjects

Morbidelli, M.

Publications and source records attributed to Morbidelli, M..

4 recordsLinked to original sources

bioRxiv2026

Closed, Automated CAR-T Cell Manufacturing: from Research to Point of Care production.

Background: Point-of-care manufacturing of CAR-T cells could reduce cost and improve patient access, but few closed, automated systems support both flexible process development and GMP production. Methods and results: We describe LimCORE, a single-use consumable for cell incubation and processing, implemented in LimGROW (manual system) and LimONE (closed and automated system). In LimGROW, T cells expanded 246 +/-18 fold over 10 days at optimized seeding density, versus 89 +/-9 fold in a G-Rex control, with similar viability (>90%) and phenotype. Process parameters directly transferred to LimONE, where automated, closed buoyancy-based CD3+ selection achieved 90.3 +/-3.5% purity and 59.2 +/-17.0% recovery in under two hours. Using this workflow, we manufactured CD19 CAR-T cells in a fully closed, automated 7-day LimONE process requiring 155 minutes of operator intervention. LimONE manufactured CAR-T cells reached 42.6 +/-2.4 fold expansion (532 +/-29x10e6; cells at harvest), with 43.6 +/-6.0% transduction efficiency and viability similar to G-Rex controls. CD4+/CD8+ ratios, CD8+ differentiation subsets, and cytotoxicity were comparable between platforms, while LimONE-manufactured cells showed increased spare respiratory capacity and oxygen consumption. Across four manufacturing runs on the LimONE, no hardware or software failures occurred, and automated liquid transfers and volume concentration stayed within +/-5% accuracy. Conclusions: These data show that LimCORE supports T cell expansion, selection, and complete CD19 CAR-T manufacturing with performance similar to existing platforms while reducing operator time, across R&D and GMP scale closed-system formats.

bioengineering↗

bioRxiv2026

Structural conservation and expanded functionality of hyper-stable human serum albumin variants

Human serum albumin (hSA) is the most abundant protein in human plasma, and its pharmacological properties, such as long plasma half-life mediated by the neonatal Fc receptor (FcRn) and its ability to bind endogenous and exogenous molecules, make it attractive for biotechnological applications. Currently, most wild type (WT) SAs are derived from human or bovine serum or produced in yeast and mammalian cells. Although well established, these methods are costly, difficult to reproduce, and not environmentally sustainable. Building on a previous study to design highly mutated hSA sequences, we extend the validation through an in-depth analysis of three engineered hSA variants; hSA1, hSA2, and hSA3, containing 16, 25, or 73 amino acid substitutions, respectively. These variants were designed for enhanced solubility, stability, and expression in Escherichia coli. All three variants showed low- micromolar affinities for hFcRn at pH 5.5, and negligible binding at pH 7.4. In a human endothelial cell-based recycling assay (HERA), the engineered hSA variants were recycled by hFcRn to the same extent as hSA isolated from serum. Exploring the properties of canonical drug-binding sites, warfarin affinity was comparable to WT hSA, whereas ibuprofen binding differed. Complementary cytotoxicity assays on human macrophages confirmed negligible toxicity and biocompatibility. A cryo-electron microscopy structure of hSA3 revealed that, despite extensive engineering, the native heart-shape of hSA, folding of domains, and its open conformation were preserved. These findings validate the structural integrity and functional adaptability of engineered hSA variants, underscoring their potential as versatile, animal-free solutions for next-generation therapeutics and biotechnological applications.

biochemistry↗

bioRxiv2026

Quicklime-based eradication attempt of Xenopus laevis as a model for controlling pondscape invasions

Aquatic non-native invasive species are notoriously difficult to eradicate, particularly in pondscapes where populations can spread rapidly, persist in unmanaged refugia, and recolonise treated sites. In such contexts, high-intensity management interventions may be justified, balancing short-term collateral impacts against the prevention of permanent establishment and long-term damages. Chemical eradication methods, such as rotenone or herbicide application, can be effective but raise ethical and environmental concerns. Here, we evaluate quicklime (calcium oxide, CaO) application as a more sustainable alternative control tool for pondscape invaders compared to other chemical methods, using the African clawed frog (Xenopus laevis) invasion in Belgium as a case study. When applied to water, quicklime hydrates exothermically to calcium hydroxide (Ca(OH)), which releases OH ions upon dissolution, temporarily and rapidly increasing pH to lethal levels. In winter 2023, three ponds with breeding populations of X. laevis of low ecological value were drained, fenced, and treated with quicklime. Treatment effectiveness was assessed through pH measurements, visual surveys, and environmental DNA (eDNA) quantification. Immediately after treatment, large numbers of deceased post-metamorphic individuals were recovered, indicating treatment-induced mortality. Eight weeks post-treatment, eDNA concentrations were markedly lower in two of the three ponds (reductions of 100% and 80%) compared to those during the same period one year later. Although eDNA concentrations increased again during the following summer suggesting partial population recovery through survival and/or recolonisation, they remained lower than pre-treatment conditions. Water pH returned to near baseline levels within one month. We provide the first field-based preliminary evidence that quicklime can induce large-scale mortality in X. laevis populations in small to medium-sized ponds. We discuss practical considerations, limitations, and broader applicability, proposing quicklime as a high-intensity option within integrated management strategies for pondscape invaders.

ecology↗

bioRxiv2025

From plasmid sequence to process design: A computational analysis of metabolism in the context of plasmid DNA manufacturing

Detailed understanding of plasmid-host physiological interactions and recombinant protein expression is crucial for the design and optimization of plasmid DNA (pDNA) manufacturing processes. Successfully transformed host cells carrying one or more copies of a particular plasmid exhibit modified metabolic behavior which may include reduced specific growth rate, increased metabolic resource cycling (ATP/ADP, NAD/NADH, NADP/NADPH) and, in some cases, even modified nutrient uptake and metabolite secretion rates. However, to the extent of our knowledge, there have been no attempts to map the impact of plasmid design directly to critical process parameters (CPPs) such as the specific growth rate and/or productivity. Herein we present, a comprehensive computational analysis based on carbon constrained Flux Balance Analysis (ccFBA) to precisely calculate the metabolic burden imposed by plasmid replication and recombinant protein expression. Explicit stoichiometric coefficients for nucleotides and amino acids were derived directly from the plasmids sequence and were used to formulate biosynthetic reactions tailored to the components of individual plasmids. Three tunable parameters were introduced to map the impact of promoter strength, copy number and choice of selection marker on the host cells metabolism. Literature derived experimental data from E. coli cultures producing three different plasmids were used to constrain the iJR904 Genome Scale Model (GeM). Our analysis revealed correlations between cellular growth rate, promoter strength and pDNA productivity with significant implications for the design of recombinant technology based manufacturing processes.

systems biology↗