Production of modified crosslinked cell‐free hemoglobin for human use: the role of quantitative determination of endotoxin contamination

Abstract

In vivo toxicity remains a major barrier to the successful use of cell-free hemoglobin (Hb) as an oxygen carrier in humans. Bacterial endotoxin (lipopolysaccharide, LPS) is known to contribute to the in vivo toxicity of Hb preparations, and the prevention of LPS contamination is a critical aspect of the effort to create an efficacious Hb blood substitute. Limulus amebocyte lysate assays for endotoxin were performed on multiple Hb samples from 26 independent production runs for the preparation of human crosslinked cell-free hemoglobin (alpha alpha Hb). High levels of LPS contamination (1- > 100 ng/mL) of alpha alpha Hb solutions were detected in multiple samples during many of the initial production runs. It was observed that LPS contamination of alpha alpha Hb solutions could occur at any step during the production sequence. Substantial enhancement by alpha alpha Hb of the biologic effects of LPS was demonstrated by two independent assays for endotoxin (the Limulus amebocyte lysate test and a mononuclear cell procoagulant assay), whereas LPS biologic activity was only slightly increased by human serum albumin and substantially diminished by IgG. These results suggest that the prevention of LPS-related toxicities in vivo may be more important to the clinical use of Hb solutions than to the use of other intravenous protein products. Therefore, it was encouraging to note that, with the careful monitoring for LPS in the production facility and in multiple samples during cell-free Hb production, sources of LPS contamination were recognized and the appropriate sites were made endotoxin-free.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

“…In previous publications, considerable increases of the biological activities of LPS in the LAL test (8,11,12,35), production of mononuclear cell tissue factor (35), and stimulation of endothelial cell tissue factor production (36, 37) as a result of Hb binding to LPS were described. Furthermore, an increase of endotoxin binding to endothelial cells and increased lethal toxicity were reported (13,36).…”

Detection of lipopolysaccharide in hemoglobin‐vesicles by Limulus amebocyte lysate test with kinetic–turbidimetric gel clotting analysis and pretreatment of surfactant

“…Bacterial LPS is an amphiphilic gigantic macromolecule, therefore, it hydrophobically interacts with protein and biomembranes 20. Hb strongly interacts with LPS, showing synergistic toxicity 21–23. The constituent of endotoxin that causes LAL gelation is a glycophospholipid that is designated lipid‐A 24.…”

Hemoglobin increases mortality from bacterial endotoxin

“…Our laboratory has demonstrated that Hb forms stable complexes with a wide variety of LPSs and that LPS macromolecular particles become disaggregated after binding to Hb (11,12). In a series of in vitro studies, Hb was shown to enhance the ability of LPS to activate coagulation (11,25), stimulate procoagulant activity from peripheral blood mononuclear cells (25), and stimulate production of procoagulant activity by cultured human endothelial cells (23). LPS binding to endothelial cells was increased when LPS was complexed with Hb (24), suggesting a possible mechanism for the enhanced cellular response to LPS-Hb complexes compared to LPS alone.…”