(162ao) Diverse Oxygen Therapeutic Applications of Surface Camouflaged Extracellular Annelid Mega-Hemoglobin

Motivation: The newest generation of hemoglobin-based oxygen carriers (HBOCs) has found generic applications as oxygen therapeutics apart from their traditional use as resuscitative fluids in transfusion medicine. Recently, HBOCs have been used in organ and graft preservation, ex-vivo machine perfusion, cell growth media supplements, hypoxic tumor normalization, and wound healing. These diverse applications have necessitated the development of oxygen therapeutics that can be used for multiple applications. Steering away from traditional mammalian hemoglobins, this work focuses on the mega-hemoglobin (LtEc) of the annelid Lumbricus terrestris for use as an oxygen therapeutic. Its large size (~30 nm) reduces the possibility of in vivo extravasation and scavenging of endothelial-derived nitric oxide (NO). The large molecular diameter of LtEc along with its’ unusually high protein stability, low rate of auto-oxidation, moderate colloid osmotic pressure, and human blood-like oxygen binding properties make it an ideal candidate for use as a resuscitative fluid in trauma treatment. Subsequently, LtEc is also a promising candidate for use in graft preservation and as an oxygen supplement in cell culture media because of its ability to stay in the reduced form for long periods of time. However, to mask the exterior protein surface and reduce potential immunogenic activity, surface conjugation is necessary. Polyethylene glycol (PEG) is a widely used and FDA approved polymer used to mask the surface of materials and improve circulatory life. We devised methods to obtain ultra-pure LtEc and surface camouflage it with PEG using thiol-maleimide chemistry. Comprehensive in vitro characterization and preliminary in vivo studies show promise for further investigation of PEG-LtEc as an oxygen therapeutic.

Methods: Purification of LtEc from Lumbricus terrestris worms was performed by modifying previously devised protocols from our lab with a focus on improving the purity of the resulting product. In short, Canadian nightcrawlers (Lumbricus terrestris) were rinsed to remove dirt and mucus prior to homogenization in a modified Tris buffer. The homogenate was centrifuged via multiple cycles to eliminate worm debris and dirt to obtain a cloudy red supernatant. Post vacuum filtration, multi-step tangential flow filtration through 0.65 µm and 0.22 µm hollow fiber filters was performed to eliminate large aggregates and bacterial components. Continuous volume diafiltrations were carried out over a 500 kDa filter to eliminate a majority of the unwanted impurities. Retentate and permeate samples were analyzed using analytical size exclusion chromatography (SEC) to track loss of proteins and the purification was concluded when the LtEc in the retentate reached >99% purity. In vitro biophysical characterization included dynamic light scattering (DLS), size exclusion high pressure liquid chromatography (HPLC-SEC), oxygen equilibrium analysis, rapid deoxygenation kinetics, gel electrophoresis (SDS-PAGE), and auto-oxidation kinetics. Surface modification of purified LtEc was carried out using thiol-maleimide chemistry by converting surface lysine residues to thiols and attaching PEG chains with monofunctional ends. Unreacted components were removed via tangential flow filtration on a 500 kDa filter, and the PEG-conjugated product was analyzed using amino acid residue assays, DLS, HPLC-SEC, and SDS-PAGE to confirm PEG modification. Oxygen equilibrium properties and other in vitro properties were tested and compared to unmodified material. The degree of surface camouflage was engineered by exploiting the numerous lysine residues on the LtEc surface and desirable in vitro properties were obtained.

Results: The modified purification protocol was successful in obtaining an LtEc product with >99% purity, which was verified using HPLC-SEC and SDS-PAGE analyses. This was an important result as the removal of non-mammalian proteins and impurities is crucial for a pharmaceutical product to be used in transfusion medicine. Biophysical characterization was performed to determine the beneficial properties of LtEc such as its’ large hydrodynamic diameter, moderate oxygen affinity, high cooperativity, low auto-oxidation rate, and fast deoxygenation kinetics. In vivo analysis in a golden Syrian hamster model corroborated that the ultra-pure product performed better in animals than product purified using older protocols with significantly better flow in arterioles and higher functional capillary density. Various degrees of surface modification with PEG resulted in PEG-LtEc products with varying biophysical properties. Even though the hydrodynamic diameter varied little, we observed changes in molecular masses of subunits using the HPLC-SEC, SDS-PAGE, and matrix-assisted laser desorption ionization (MALDI) mass spectral analyses, further confirming successful surface camouflage. The attachment of PEG resulted in a product with higher oxygen affinity, lower cooperativity, higher auto-oxidation rates, and slower kinetic rate of oxygen offloading compared to unmodified LtEc. In spite of these changes to the biophysical properties of PEG-LtEc, it is expected that future in vivo evaluation of the PEGylated products will yield longer circulatory times and reduced immunogenic responses compared to unmodified LtEc.

Takeaways: The streamlined LtEc purification protocol devised in the study allows for the production of an extracellular hemoglobin with >99% purity. This purity is essential to enable a wide variety of applications of LtEc as an oxygen therapeutic. We show varying extents of camouflage of the LtEc surface with PEG and its’ effect on the biophysical properties of the product. PEG-LtEc is expected to have longer circulation time in vivo compared to unmodified LtEc due to its surface camouflage. Future animal studies will test this hypothesis. However, the use of LtEc can be expanded to cell media supplement and graft preservation, making it a diverse oxygen therapeutic.