Purification of hemoglobin by tangential flow filtration with diafiltration

Abstract

A recent study by Palmer et al. (2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) & 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three-stage TFF process is sufficient to produce HPLC-grade bHb.

Tissue oxygenation after exchange transfusion with ultrahigh-molecular-weight tense- and relaxed-state polymerized bovine hemoglobins

“…T-state PolybHb was synthesized according to previously described methods (5,12). To generate fully deoxygenated (T-state) bHb, 30 g of purified bHb were diluted with phosphate buffer (20 mM, pH 8.0) to yield 1,200 ml of bHb solution.…”

“…Fresh bovine blood collected in 3.8% sodium citrate solution at a final concentration of 90:10 (vol/vol) (bovine blood-sodium citrate solution) was purchased from Quad Five (Ryegate, MO). bHb was purified from lysed bovine RBCs (bRBCs) via tangential flow filtration (12,31). bRBCs were initially washed three times with 3 volumes of isotonic saline solution (0.9%) at 4°C.…”

Biophysical Properties and Oxygenation Potential of High-Molecular-Weight Glutaraldehyde-Polymerized Human Hemoglobins Maintained in the Tense and Relaxed Quaternary States

“…[18][19][20] PolyhHb synthesis T-state PolyhHb was synthesized as previously described in the literature. 9,[21][22][23] hHb was diluted with 20 mM phosphate buffer (PB; pH 8.0) to yield 1.5 L of 0.3 mM hHb that was stored in a 2 L glass bottle submerged in an ice bath.…”

Functional comparison of hemoglobin purified by different methods and their biophysical implications

“…However, an ample supply of purified hemoglobin (Hb) is needed to synthesize HBOCs and meet the growing world-wide demand for RBC transfusions (Basu, 2003;Reiss, 2001). Typical methods that have been developed to purify Hb include ion exchange chromatography (IEC, Sun and Palmer, 2008) and tangential flow filtration (TFF, Elmer et al, 2009;Palmer et al, 2009). An ideal strategy for purifying Hb should remove viruses and endotoxin, as well as other protein impurities that could influence HBOC product safety.…”