An enhanced high-yield manufacture process for engineered recombinant Hemoglobin-Based Oxygen Carriers for oxygen therapeutics and blood substitutes.

Context: Artificial hemoglobin-based oxygen carriers (HBOCs) have long had potential in transfusion medicine due to their universal compatibility, sterility, stability, and ability to transport oxygen to tissues. Current research views them as an 'oxygen bridge' facilitating diffusion of oxygen from red blood cells to hypoxic tissues even when circulation is failing, helping protect vital organs. Previous generations of HBOCs primarily used chemical modifications to stabilise hemoglobin (Hb) outside of blood cells, but this approach did not address the issue of the inherent cytotoxic chemistries of Hb. We have utilised genetic engineering to create a synthetic HBOC that is the first to tackle issues of oxidative damage and nitric oxide scavenging by re-engineering the fundamental properties of the Hb molecule, while also focusing on product stability. Our novel HBOC resolves the inherent toxicities of cell-free Hb that caused adverse side effects in previous generations of HBOCs. We have developed a uniquely homogenous PEGylation method product that preserves the HBOC’s ability to cooperativity oxygen binding. Coating the HBOC with polyethylene glycol (PEG) extends its lifespan in the bloodstream, increases viscosity, and decreases its immunogenicity. The Challenge: Previous MRC DPFS funding allowed us to successfully develop, manufacture, and test the product in focused pre-clinical trials, but yields of protein were too low to be commercially attractive. This proposal aims to address that gap, de-risking the investment by developing a high-yield production method, making the product economically viable and attractive for future investment. We are partnering with Ultrox Ltd, a commercial company. They are drafting the first Target Product Profile (TPP) and will attract venture capital funding and lead a comprehensive pre-clinical program and Phase I/II clinical trials. They have built a network of international expertise to address the manufacturing challenge and have recruited scientific advisers from Baxter Therapeutics (previously Somatogen) who worked on an earlier project to produce a recombinant HBOCs (rHb) at industrial scales between 1990-2005. That project ultimately failed in clinical trials due to instability and associated inflammatory responses, issues we have addressed in our current product. However, Baxter's manufacturing program achieved yields much higher than typical rHb production methods and the scientists who worked on the Baxter project have assisted us in enhancing our current production process, resulting in more than a tenfold increase in production yields in initial tests. Ultrox have also conducted horizon scanning to identify a leading innovator with expertise in protein and plasmid DNA production (enGenes Biotech). We plan to sub-contract that element of the project to them. Aims and Objectives: This application seeks funding to refine a high-yield method of E.coli fermentation for product manufacturer to generate our rHb on scales not seen since the Baxter product development and to confirm that changes to upstream manufacture do not negatively affect product quality and efficacy. Applications and benefits: An approved HBOC would have a global impact in healthcare. Clinical markets for our product will include trauma, stroke, myocardial infarction, shock, sepsis, sickle cell crisis and organ transplantation. Patients requiring red blood cells who are immunocompromised and those that refuse blood components due to religious reasons can also benefit from a synthetic HBOCs. Addressing this gap in manufacturing is essential given the needs for bulk Hb production in an HBOC product. It will have knock-on advantages for other scientists generating recombinant hemoproteins for research and clinical applications.