Tangential flow filtration of haptoglobin

Abstract

Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 μm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cutoffs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.

Purification and analysis of a protein cocktail capable of scavenging cell‐free hemoglobin, heme, and iron

“…Purification of the protein cocktail followed the TFF Hp purification method with the addition of one additional TFF stage at the end of the original process 18 . Briefly, 500 g of FIV was suspended in 5 L of phosphate‐buffered saline (PBS) and homogenized in a blender.…”

“…Samples from the purification process were separated via HPLC‐SEC using a commercial column (4.6 × 300 mm Acclaim SEC‐1000, Thermo Fisher Scientific, Waltham, MA) attached to an HPLC system (Dionex UltiMate 3000, Thermo Fisher Scientific, Waltham, MA) as described previously in the literature 18 …”

“…In a previous study, we demonstrated the purification of Hp via tangential flow filtration (TFF) of Human Cohn Fraction IV (FIV) using a series of hollow fiber filters to bracket the plasma proteins based on their molecular weight (MW) 18 . The key advantages of this process are that TFF is an easily scalable separation method and FIV is a waste product from the plasma fractioning industry, making the Hp purification process cost‐effective and amenable for large‐scale Hp production.…”

“…The key advantages of this process are that TFF is an easily scalable separation method and FIV is a waste product from the plasma fractioning industry, making the Hp purification process cost‐effective and amenable for large‐scale Hp production. Interestingly, based on the composition of FIV, the last permeate fraction from the TFF Hp purification process may serve as a source of the therapeutic proteins required for comprehensive hemolysis treatment 18 . In this study, the last permeate of the TFF Hp purification process was retained on a 50 kDa hollow fiber (HF) filter, and the isolated product was characterized to develop a cost‐effective and simple protein cocktail for potential treatment of hemolysis.…”

Apohemoglobin-haptoglobin complexes attenuate the hypertensive response to low-molecular-weight polymerized hemoglobin

“…The Hp used for this study was purified from human Cohn fraction IV derived from pooled human plasma. 31 The resulting Hp contained both Hp2-1 and Hp2-2 phenotypes. The total amount of protein in solution was estimated by using a Bradford assay.…”