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. Materials and Methods We used tangential flow filtration (TFF), an easily scalable and affordable technique, to produce highly pure LtEc from Earthworms. The purity, yield, methemoglobin level, viscosity, colloid osmotic pressure, O2 binding equilibria, and ligand binding kinetics of the purified LtEc was measured in vitro. The purified LtEc product was then evaluated in hamsters using a hypervolemic infusion model to establish its basic biocompatibility and detect any changes in microcirculatory and systemic parameters. Results TFF was able to produce LtEc with high purity and yield (5–10 grams per 1000 worms). The purified LtEc product did not elicit hypertension or vasoconstriction when infused into hamsters. Conclusion LtEc may be easily purified and safely transfused into hamsters in small amounts (0.5–1.5 g/dL final concentration in blood) without any noticeable side-effects. Therefore, LtEc may serve as a very promising oxygen carrier for use in transfusion medicine.
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.
CART therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19 + B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CART cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time-consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non-viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non-viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.
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.
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