Internal insertion in albumin improves fusion-protein integrity while maintaining bioactivity and FcRn interaction

Human serum albumin (HSA) is a clinically validated carrier for half-life extension via FcRn recycling, yet conventional N- or C-terminal albumin fusions can compromise fusion-partner activity or increase product heterogeneity. Here we evaluated an internal insertion between HSA residues 363 and 364 using two fusion proteins with distinct sizes and termini geometries: interleukin-2 (IL2, 15.5 kDa) and soluble TNFR2 (sTNFR2, 25 kDa). In HEK293F cells, internal insertion produced predominantly monomeric fusion proteins with reduced fragmentation compared to the N-terminal fusions based on gel filtration and SDS-PAGE profiles. The IL2 internal-insertion fusion (I-IL2) retained activities of binding to CD25-positive cells and stimulating cell proliferation (EC 50  = 9.50 nM). sTNFR2 internal-insertion fusions retained TNFα binding with nanomolar apparent affinities by surface plasmon resonance, while another internal fusion flanked with linkers showing the strongest binding (apparent K D  = 9.52 nM) and the highest neutralizing potency in the activity assay (EC 50  = 3.0 nM). For the IL2 fusion, FcRn binding at pH 6.0 was found to be in the micromolar range, with I-IL2 showing stronger apparent binding than N-IL2 under identical assay conditions. Molecular modeling and structural overlays suggested that the inserted domains for all fusions are spatially separated from the FcRn-binding surface of albumin while remaining accessible for receptor binding. Together, these results identify HSA 363–364 as a promising internal insertion site that preserves fusion-partner function.