IntroductionHuman serum albumin (HSA) is an essential plasma protein with widespread applications in medicine and biotechnology. It functions as a stabilizer in vaccine formulations, a therapeutic agent in conditions such as severe burns and hemorrhagic shock, and a vital supplement in serum-free cell culture media1. The global demand for HSA exceeds 500 metric tons annually, largely fulfilled through plasma fractionation. However, this method is constrained by limited donor availability, high production costs, and potential biosafety risks associated with blood-borne pathogens2. To address these limitations, alternative recombinant expression platforms including microbial, yeast, and mammalian cell cultures have been explored. Yet, each presents inherent challenges: Escherichia coli lacks post-translational modifications (PTMs) machinery critical for protein activity3, while yeast and mammalian systems require complex culture conditions, bioreactor infrastructure, and long production cycles, driving up costs and complexity4,5,6. These challenges underscore the pressing need for more scalable, cost-effective, and sustainable production systems for biopharmaceuticals.Plant molecular farming has emerged as a versatile and increasingly validated platform for recombinant protein production. It offers several advantages, including lower production costs, rapid scalability, intrinsic biosafety, and the capacity for PTMs6,7. Among transient expression systems, Nicotiana benthamiana has become a workhorse due to its susceptibility to Agrobacterium tumefaciens-mediated transformation and its amenability to scalable agroinfiltration techniques8,9,10. Transient expression enables high-level protein production within days, without requiring stable transformation or tissue culture. Previous efforts have demonstrated the feasibility of expressing recombinant HSA (rHSA) in plants4,11,12, but few studies have systematically optimized expression parameters or assessed protein functionality alongside environmental sustainability, leaving critical gaps in process development for plant-based rHSA production.In addition to production efficiency, sustainability and regulatory compliance are key considerations in biomanufacturing. Plasma-derived and mammalian cell-based HSA productions involve significant water, energy, and chemical inputs, contributing to high environmental footprints13,14. Even microbial systems require energy-intensive fermentation and generate considerable downstream waste15. While plant-based systems are often considered greener alternatives, quantitative assessments of their environmental performance remain scarce. Sustainability metrics such as solvent efficiency, energy consumption, waste-to-product ratio (WPR), process mass intensity (PMI), the Green Analytical Procedure Index (GAPI), and its extension, the Complex Modified GAPI (ComplexMoGAPI), provide structured frameworks for evaluating solvent usage, reagent toxicity, energy demands, and waste generation throughout both production and analytical workflows16,17. Applying these tools to plant-based recombinant protein production can yield critical insights to guide both process improvement and environmental benchmarking.This study aims to optimize the transient expression of rHSA in N. benthamiana by systematically evaluating agroinfiltration parameters, including days after infiltration, bacterial optical densities, and plant age, to maximize protein yield. The biological functionality of purified protein is validated using NIH3T3 fibroblast proliferation assays, and a comprehensive sustainability assessment evaluation is conducted using the ComplexMoGAPI flamework. Matrices including solvent use, energy consumption, and waste-to-product ratio are quantified to identify key environmental challenges and opportunities. By integrating biotechnological and sustainability perspectives, this study contributes to the growing body of work advancing plant molecular farming as a commercially viable and environmentally responsible solution for producing essential recombinant proteins such as HSA. Importantly, the findings also support the future regulatory potential of plant-derived biotherapeutics by demonstrating product integrity, functional equivalence, and a lower ecological impact—key criteria in emerging biosafety and sustainability guidelines18,19.ResultsOptimization of rHSA expression efficiency in N. benthamianaIn the present study, specific infiltration parameters were systematically assessed to identify optimal conditions that enhance rHSA protein yield in N. benthamiana. The pBYR2e geminiviral vector and Agrobacterium tumefaciens strain GV3101 were used for transient expression in planta.Effect of days post-infiltration (dpi) on rHSA accumulationTo determine the optimal harvest time for rHSA production, leaves were collected at 2, 4, 6, 8, and 10 dpi, and protein expression levels were quantified using Western blot densitometry (Fig. 1A and Supplementary Fig. S1). The results revealed a time-dependent increase in rHSA accumulation, reaching a peak at 4 dpi. Expression levels remained relatively high at 6 dpi but declined significantly after 8 dpi (p