During 1990 and 1991 the capability for repetitive, consecutive production of DCLHb solution to meet a rigorous and complete set of product criteria was demonstrated. In addition, through periodic monitoring of product stored under controlled conditions, the stability of all lots of DCLHb solution during frozen storage was demonstrated for more than a year. In this way, assurance was provided that the DCLHb solution used in preclinical testing met all product criteria throughout the biological testing period.

During the course of our studies it became clear that there were therapeutic applications for which a polymeric hemoglobin having an extended half-life in circulation would be appropriate. Therefore, a process for the glutaraldehyde-polymerization of diaspirin cross-linked hemoglobin (DCLHb) was developed and used to prepare glutaraldehyde-polymerized DCLHb (GP-DCLHb) in lactated Ringer's solution in sufficient quantities for biological testing. Both isovolemic exchange-transfusion and "top-load" studies (rats; primates and swine, respectively) were completed in which a broad spectrum of physiologic, histopathologic and analytical parameters were monitored and assessed. In general, GP-DCLHb in lactated Ringer's solution was well-tolerated physiologically. When compared to DCLHb, GP-DCLHb offers the advantages of reduced renal clearance of hemoglobin and an extended half-life in circulation. GP-DCLHb has the disadvantages that (1) glutaraldehyde is an ineffective virucidal agent under the conditions of the polymerization reaction and a separate virus inactivation step is required; (2) low-endotoxin (LAL-negative) GP-DCLHb solutions are pyrogenic (rabbits); and (3) unusual deposition of hemoglobin-containing material in the small arterioles of the liver and kidney (rats) was sometimes seen even after a period of time (2 weeks) during which treatment-related organ pathologies are usually resolved, a finding peculiar to GP-DCLHb among the various hemoglobin derivatives we have tested.

Diaspirin cross-linked hemoglobin (DCLHb), a human hemoglobin that is intramolecularly cross-linked between the alpha chains (lysine 99(alpha)(1)-lysine 99(alpha)(2)), was polymerized with a number of water-soluble, nonimmunogenic polyamide cross-linking agents. The degree of polymerization and the oxygen-carrying capacity depended upon the polyamide reagent, the starting concentration of DCLHb, the molar ratio of the polyamide reagent to DCLHb used, the reaction pH, and whether oxy- or deoxy-DCLHb was used in the polymerization reaction.

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