Small scale purification and biophysical characterization of haptoglobin derived from human Cohn fraction IV.

Haptoglobin (Hp) is a polymorphic acute phase α-2 glycoprotein found in plasma that plays a critical role in binding, neutralizing, and removing cell-free hemoglobin (Hb) from the circulation. Under clinical conditions characterized by high levels of hemolysis, such as in patients with sickle cell disease (SCD), large quantities of cell-free Hb are released from lysed red blood cells (RBCs) into the circulation and bind to Hp. This interaction reduces the plasma Hp concentration below basal levels and diminishes its Hb-binding capacity. Therefore, plasma-derived Hp has the potential to be used therapeutically to scavenge, neutralize, and remove excess cell-free Hb from the blood, thus preventing Hb-mediated toxicity. This provides strong motivation to purify Hp at high purity using sustainable sources, such as waste plasma fractions from the Cohn plasma fractionation process used to produce human serum albumin from pooled plasma. Starting from human Cohn fraction IV, we first enriched an Hp-rich fraction using tangential flow filtration (TFF), which was then used as the starting material for purification. Hp was subsequently purified from this fraction using hydrophobic interaction chromatography (HIC) to homogeneity. The final Hp purity reached 98% by SDS-PAGE densitometry and 87% by trypsin digest LC-MS/MS analysis. The purified Hp was further characterized to determine its molecular weight, secondary structure, Hb-binding capacity, and binding kinetics using biophysical techniques including MALDI-TOF, circular dichroism, size exclusion HPLC, and UV-visible stopped-flow spectroscopy. More importantly, the simplicity and efficiency of the TFF-HIC workflow demonstrated strong potential for scalable Hp production.