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Purification of hemoglobin by tangential flow filtration with diafiltration.

ABSTRACT: A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 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. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 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.

SUBMITTER: Elmer J

PROVIDER: S-EPMC2783993 | biostudies-other | 2009 Sep-Oct

REPOSITORIES: biostudies-other

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Purification of hemoglobin by tangential flow filtration with diafiltration.

Elmer Jacob J Harris David R DR Sun Guoyong G Palmer Andre F AF

Biotechnology progress 20090901 5

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 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. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and ...[more]

PMID: 19621471

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Project description:Background and objectivesRed blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump.Materials and methodsThe TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis.ResultsNo substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12.ConclusionOverall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Project description:Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

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Purification of hemoglobin by tangential flow filtration with diafiltration.

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Purification of hemoglobin by tangential flow filtration with diafiltration.

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