Plasma fractionation unused intermediates proteome as a potential source of new therapies for ultra-rare diseases

Human plasma, characterized by a highly complex proteome, represents an important resource for developing therapeutics targeting rare diseases caused by plasma protein deficiencies. Despite this complexity, only a limited number of plasma-derived proteins are currently used in approved therapies. At the same time, industrial plasma fractionation processes generate substantial quantities of unused intermediates that remain largely unexplored but may represent valuable and cost-effective sources of biologically active proteins. Their characterization could support the discovery of novel therapeutics while improving plasma utilization. Plasma fractionation workflows across four Kedrion manufacturing sites (Bolognana, Italy; Melville, USA; Godollo, Hungary; and Elstree, UK) were investigated, focusing on unused intermediates. Solubilization conditions were optimized based on protein content and patterns to enable proteomic analysis by LC-MS/MS. In parallel, a semi-quantitative, high-throughput aptamer-based proteomics platform was applied. This complementary approach validated and extended LC-MS/MS findings, increasing dataset robustness. Comparative proteomics and bioinformatics analyses enabled comprehensive mapping of protein composition across sites. From this dataset, plasminogen (PLG) was selected as a candidate for therapeutic development using unused intermediates. PLG plays a key role in fibrinolysis, wound healing, and tissue remodeling. Currently, the only approved indication for PLG therapy is Type I deficiency, with treatment produced from whole plasma using a dedicated process with limited scalability. Using fractionation intermediates could enable more sustainable production and expand availability for additional indications such as chronic wounds and burns. The presence and suitability of PLG in unused intermediates were then confirmed. Proteomic profiling was followed by validation through western blot and antigen-based assays. Functional characterization guided the selection of optimal material and conditions. The first filtration residual from cold ethanol precipitation of cryo-supernatant at the Bolognana site (Fraction I) was identified as the best starting material. Solubilization strategies were optimized to maximize recovery of native Glu-PLG, enabling development of an affinity chromatography purification workflow. The experimental PLG was characterized for purity, content, and activity and benchmarked against a reference control. The prototype achieved approximately 87% purity versus 100% for the control, while maintaining comparable specific activity and Glu-PLG content, demonstrating proof of concept. Functional activity of the experimental PLG was evaluated in comparison with the reference control. A plasmin generation assay assessed fibrinolytic capacity, a clot lysis time assay quantified fibrin degradation, and an in vitro scratch assay with A549 cells evaluated cell migration. Across all assays, experimental PLG showed activity comparable to the control, supporting plasmin generation, fibrinolysis, and cell migration. These findings demonstrate functional equivalence between preparations. Overall, this work shows that unused plasma fractionation intermediates are a valuable source of therapeutic proteins. The development of an experimental PLG prototype highlights the potential to expand plasma utilization and enable cost-effective production of new plasma-derived therapies.