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

Area of Science:

• Biochemistry
• Protein Chemistry
• Pharmaceutical Manufacturing

Background:

• Hemopexin (Hx) is a plasma glycoprotein that binds free heme.
• Hx is being developed as a therapeutic agent.
• Solvent/detergent (S/D) treatment is standard for viral inactivation in plasma-derived products.

Purpose of the Study:

• To investigate the structural impact of S/D treatment on Hx.
• To determine if elevated ionic strength can mitigate S/D-induced perturbations.
• To ensure Hx structural integrity and function during manufacturing.

Main Methods:

• Size-exclusion HPLC (SE-HPLC) to assess apparent size.
• Functional assays to measure Hx activity.
• Small-angle X-ray scattering (SAXS) to analyze molecular envelope changes.

Main Results:

• S/D treatment reduced Hx activity and increased its hydrodynamic size.
• SAXS revealed S/D-induced expansion of the Hx molecular envelope.
• Increased ionic strength (NaCl) counteracted S/D effects, restoring native state and activity.

Conclusions:

• Hx is susceptible to structural disruption by S/D treatment, unlike many plasma proteins.
• Ionic strength modulation is an effective strategy to maintain Hx structural integrity and function.
• This finding is crucial for the safe and effective manufacturing of Hx therapeutics.