Tetrameric Modified Hemoglobins

Two intramolecularly crosslinked hemoglobin tetramers - DCLHb and rHbl.1 - stand out from among those in a cornucopia of hemoglobin derivatives that were prepared at laboratory scales and screened in the late twentieth century. Each of these proteins was designed to meet then-current requirements for 'blood substitutes'. For example, it was widely postulated that each would be a replacement for transfusion of packed red cells, and provide both colloidal volume expansion and oxygen-delivery characteristics similar to those exhibited by fresh whole blood. Likewise, stringent purification and incorporation of an internal crosslink to prevent dissociation of cell-free hemoglobin into dimers were goals that would, it was believed, address still other shortcomings of unmodified acellular hemoglobin.

At the time, preparation of a stabilized Hb tetramer modeled after human Hb appeared to be reasonable and sufficient to meet the postulated requirements for an 'artificial blood'. It was believed that a tetramer would be sufficiently large to lengthen the circulating half-life and prevent the hemoglobinuria and renal injury that restricted use of unmodified acellular hemoglobin. In addition, advances in protein characterization had pinpointed ways in which dissociation to dimers could potentially be eliminated by

Blood Substitutes, edited by Robert M. Winslow. ISBN-13: 978-0-12-759760-7 ISBN-10: 0-12-759760-3

incorporation of inter-subunit crosslinks, either embedded in the globin or at termini of the a-subunits, which lay in close proximity.

Likewise, prevalent assumptions concerning the optimal oxygen-binding characteristics of an acellular hemoglobin guided blood substitute development. Within the human red cell, it was recognized, oxygen release is facilitated by the binding of the polyanionic allosteric effector, 2,3-diphosphoglycerate (2,3-DPG), which stabilizes hemoglobin in the lower oxygen affinity conformation. During red cell storage, the concentration of 2,3-DPG decreases to near undetectable values, and the hemoglobin in the stored RBCs has a higher affinity for oxygen. As a consequence of the significant increase in oxygen affinity, marked by a left shift in the oxygen dissociation curve, oxygen delivery decreases from ~4.7g/dl immediately after blood collection to less than half that value about 10 days later, when the 2,3-DPG is fully depleted.These observations suggested that the oxygen-delivery characteristics of choice would comprise a P50 of about 28 mmHg (with a Hill coefficient, n, of about 2.9) and an oxygen-delivery capability of about 5 ml/dl.

To achieve these goals, chemistry or biotechnology, respectively, was developed to ensure highly specific intramolecular crosslinking. In the case of DCLHb, application of the crosslinking technology concurrently accomplished favorable alteration of the oxygen-binding function. In the case of the recombinant hemoglobins, an Asparagine 108p : Lysine mutation (i.e., the Hb Presbyterian mutation) altered the oxygen-binding function in a manner that mimicked that of fresh blood.

DCLHb and the HemAssistâ„¢ (DCLHb 10 per cent and electrolyte injection, Baxter Healthcare Corporation) dosage form in which it was incorporated are unique among other hemoglobin-based oxygen carriers (HBOCs) of the period in many respects. For example, these products provided to the clinical community consistent, injectable-quality material that was produced from human hemoglobin using rugged and reproducible, validated processes at three different manufacturing sites, including one in Europe. These properties encouraged investigators both to probe more exhaustively and to expand their research into the properties and potential therapeutic applications of acellular hemoglobin. The resulting studies of DCLHb confirmed that, in addition to its oxygen-carrying properties, Hb functions as an active oncotic and pharmacological agent. Ultimately, these same properties also contributed to its demise for applications as anything other than a research tool.

Similarly, the successful development of recombinant hemoglobin and its injectable dosage form, (Optroâ„¢, recombinant human hemoglobin 8 g/dl and electrolyte injection, Somatogen, Inc.) marks a unique achievement. The ability to prepare cell-free hemoglobin solutions using human hemoglobin synthesized in E. coli and the yeast Saccharomyces cerevisiae presented an attractive alternative to blood substitutes based on hemoglobins sourced from mammalian red cells. Further, the observation that a recombinant Hb could be modified by the techniques of protein engineering to modulate oxygen affinity and stabilize the protein from dissociation ensured a consistent supply of an oxygen-delivering protein free of blood-borne pathogens.

At the time, it was widely recognized that, in many respects, E. coli is an excellent choice of organism in which to express heteromultimeric proteins such as human hemoglobin. Nonetheless, Somatogen's decision in the early 1990s to continue development in E. coli of a recombinant hemoglobin for use as a blood substitute corresponds to near-swashbuckling bravado that, even today, flies in the face of prevailing wisdom. First and foremost, investigators had repeatedly warned against the toxicities associated with interactions between bacterial endotoxin and hemoglobin (Levin and Roth, 1998). Expression in E. coli provided molecules of recombinant hemoglobin awash in a sea of bacterial endotoxin. In addition, fermentation substituted a source containing about 95 per cent of the desired hemoglobin raw material with a source containing, at best, about 15 per cent of the desired raw material in a complex mixture of other proteins (Bunn and Forget, 1986). The ability to scale rHb fermentation and processing to commercial volumes in a reproducible manner crowns the rHb team's achievements.

The systematic manipulation of the physico-chemical properties of recombinant hemoglobins also distinguishes the rHbs from other HBOCs of the period. In particular, design and production of a polymerized rHb having a significantly reduced rate of reaction with nitric oxide and acceptable oxygen-binding properties positioned rHb2.0 for imminent clinical success. Unfortunately, the observation of an unexpected and unexplained side effect in Phase I clinical trials of these novel rHbs devastated the program and metamorphosed recombinant hemoglobins into research tools.

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