Ionic strength mitigates solvent/detergent–induced tertiary expansion of hemopexin

Hemopexin (Hx) is a plasma glycoprotein that scavenges free heme with sub-nanomolar affinity and under development as a therapeutic. Solvent/detergent treatment (S/D) is routinely applied to plasma-derived manufacturing for viral safety and generally considered non-disruptive. However, the histidine- and aromatic residue-rich heme pocket at the inter-domain interface may render Hx susceptible to S/D. We investigated whether S/D perturbs the tertiary structure of Hx and elevated ionic strength mitigates such effects. Changes in the apparent size and activity of Hx exposed to S/D were assessed by size-exclusion HPLC (SE-HPLC) and functional assays. S/D consistently reduced activity and shifted SE-HPLC profiles toward a larger hydrodynamic size. Small-angle X-ray scattering (SAXS) revealed expansion of the molecular envelope of Hx by S/D, suggesting that the heme pocket may initiate hinge relaxation between two β-propeller domains of Hx. To mitigate such expansion by S/D, we screened a formulation range of NaCl concentration. Increasing ionic strength compacted SAXS profiles toward the native state and restored activity, consistent with pocket stabilization by charge. These findings identify Hx as an exception to typical S/D tolerance and demonstrate that modulation of ionic strength represents an effective strategy to preserve structural integrity and function while fulfilling viral safety requirements.