<p>L-asparaginase is a biopharmaceutical enzyme used as a first-line treatment for acute lymphoblastic leukemia (ALL). Despite its clinical relevance, only a limited number of microbially produced L-asparaginase formulations are commercially available. Among these, PEGylated versions are preferred due to their reduced dosing frequency and lower incidence of side effects. However, PEGylated enzymes are significantly more expensive to produce, and techno-economic evaluations of such products remain scarce in the literature. This study presents a conceptual process design and a techno-economic analysis, conducted through simulation, for the production of PEGylated L-asparaginase. Based on the scale-up of process parameters developed by our research group, multiple production scenarios were evaluated. The simulation of different fermentation modes and final biomass concentration revealed key cost drivers, including biomass yield, PEGylation reagent cost, and the efficiency of unreacted enzyme removal during purification. In the best-performing scenario, which employed fed-batch fermentation with 40&#xa0;g/L of biomass, the unit production cost (UPC) of L-asparaginase was estimated at USD 5.81 per mg. Among all factors, plant depreciation and the cost of mPEG-NHS were identified as the most influential factors on the UPC. The findings support the feasibility of developing small-scale biopharmaceutical production facilities aimed at enhancing local self-sufficiency and reducing vulnerability to global supply chain disruptions.</p> Graphical Abstract <p></p>