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. The 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.