Jacob Elmer

Purification of hemoglobin by tangential flow filtration with diafiltration

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.

Hypervolemic infusion of Lumbricus terrestris erythrocruorin purified by tangential-flow filtration

BACKGROUND: The hemoglobin of the earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high‐molecular‐weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells. However, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc.

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

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (kox), responses to physiological conditions (pH, [Cl−], [IHP], and T), and ligand binding kinetics (O2, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O2 carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85.

Biophysical Properties of Lumbricus terrestris Erythrocruorin and Its Potential Use as a Red Blood Cell Substitute

Previous generations of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been plagued by key biophysical limitations that result in severe side-effects once transfused in vivo, including protein instability, high heme oxidation rates, and nitric oxide (NO) scavenging. All of these problems emerge after mammalian Hbs are removed from red blood cells (RBCs) and used for HBOC synthesis/formulation. Therefore, extracellular Hbs (erythrocruorins) from organisms which lack RBCs might serve as better HBOCs. This review focuses on the erythrocruorin of Lumbricus terrestris (LtEc), which has been shown to be extremely stable, resistant to oxidation, and may interact with NO differently than mammalian Hbs. All of these beneficial properties show that LtEc is a promising new HBOC which warrants further investigation.

Purification of Hemoglobin from Red Blood Cells using Tangential Flow Filtration and Immobilized Metal Ion Affinity Chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.

Oxygen delivery during extreme anemia with ultra-pure earthworm hemoglobin

AIM Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with high molecular weight (3.6MDa), low autoxidation rate, and limited nitric oxide (NO) dioxygenation activity. These properties make LtEc a potential candidate for use as red blood cell (RBC) substitute, i.e. Hb-based oxygen carrier (HBOC). Previous studies have shown that small amounts of LtEc can be safely transfused into mice, rats, and hamsters without eliciting major side effects. Therefore, this study was designed to understand oxygen (O(2)) transport to tissues and systemic/microvascular hemodynamics induced by LtEc during anemic conditions. MAIN METHODS Hamsters fitted with dorsal window chambers were hemodiluted to 18% hematocrit (Hct) using 6g/dL dextran 70kDa (Dex70). Hemodilution was then continued to 11% Hct using 10g/dL LtEc, 6g/dL Dex70 or 10g/dL human serum albumin (HSA). Blood pressure, heart rate, blood gas parameters, microvascular hemodynamics, microvascular blood flow, functional capillary density (FCD), intravascular pO(2) and perivascular pO(2) were studied. KEY FINDINGS LtEc maintained blood pressure without inducing vasoconstriction while increasing microvascular perfusion and FCD relative to Dex70 and HSA. LtEc increased blood O(2) carrying capacity and maintained systemic and microvascular parameters without decreasing arteriolar diameter or increasing vascular resistance with during extreme anemia. LtEc increased O(2) delivery compared to conventional plasma expanders. SIGNIFICANCE LtEc or synthetic molecules that replicate the characteristics of LtEc could be effective O(2) carriers with potential to be used in transfusion medicine to prevent tissue anoxia resulting from severe anemia.

Synthesis and biophysical properties of polymerized human serum albumin.

The use of many plasma expanders (PEs) is often limited by undesirable side effects, such as red blood cell (RBC) aggregation (hydroxyethyl starch), nephrotoxicity (dextran), and extravasation (albumin). Despite its natural prevalence in the bloodstream, human serum albumin (HSA) can increase the risk of mortality when administered to patients with increased vascular permeability (i.e., patients suffering from burns, septic shock, and endothelial dysfunction). The harmful extravasation of HSA can be limited by polymerizing HSA to increase its molecular size. In this study, HSA was nonspecifically cross‐linked with glutaraldehyde at different cross‐link densities by varying the molar ratio of glutaraldehyde to HSA. The results of this study show that the weight‐averaged molecular weight (MW), viscosity, and extent of RBC aggregation of polymerized HSA increases with increasing cross‐link density, whereas the colloid osmotic pressure (COP) decreases with increasing cross‐link density. Interestingly, circular dichroism measurements indicate that the secondary structure of HSA is unaffected by polymerization. Altogether, these results show that glutaraldehyde can effectively cross‐link HSA to produce high MW polymers, yielding a novel series of potential PEs that exhibit low COP and high viscosity.

Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases.

Direct comparison of oligochaete erythrocruorins as potential blood substitutes

Abstract While many blood substitutes are based on mammalian hemoglobins (e.g., human hemoglobin, HbA), the naturally extracellular hemoglobins of invertebrates (a.k.a. erythrocruorins, Ecs) are intriguing alternative oxygen carriers. Specifically, the erythrocruorin of Lumbricus terrestris has been shown to effectively deliver oxygen in mice and rats without the negative side effects observed with HbA. In this study, the properties of six oligochaete Ecs (Lumbricus terrestris, Eisenia hortensis, Eisenia fetida, Eisenia veneta, Eudrilus eugeniae, and Amynthas gracilis) were compared in vitro to identify the most promising blood substitute candidate(s). Several metrics were used to compare the Ecs, including their oxidation rates, dissociation at physiological pH, thermal stability, and oxygen transport characteristics. Overall, the Ecs of Lumbricus terrestris (LtEc) and Eisenia fetida (EfEc) were identified as promising candidates, since they demonstrated high thermal and oligomeric stability, while also exhibiting relatively low oxidation rates. Interestingly, the O2 affinity of LtEc (P 50 = 26.25 mmHg at 37 °C) was also observed to be uniquely lower than EfEc and all of the other Ecs (P 50 = 9.29–13.62 mmHg). Subsequent alignment of the primary sequences of LtEc and EfEc revealed several significant amino acid substitutions within the D subunit interfaces that may be responsible for this significant change in O2 affinity. Nonetheless, these results show that LtEc and EfEc are promising potential blood substitutes that are resistant to oxidation and denaturation, but additional experiments will need to be conducted to determine their safety, efficacy, and the effects of their disparate oxygen affinities in vivo.

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross‐linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from Tm = 57°C for native LtEc up to Tm = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8–10), cross‐linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross‐linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross‐linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringers Solution, which contains antioxidants. Overall, cross‐linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields.