Validation of the Heat Treatment Step Used in the Production of Diaspirln Crosslinked Hemoglobin (DCLHb™) For Viral Inactivation

Abstract

A series of experiments was performed to assess the ability of the heat treatment step used in the manufacture of diaspirin crosslinked hemoglobin (DCLHb) to inactivate viruses. In-process solutions (reaction mixtures after the crosslinking process) from six different manufacturing lots were used as test media in a 1:680 scaled down system in which the key process parameters used in the large scale production were duplicated. The inactivation of five different viruses (Bovine Viral Diarrhea Virus, Pseudorabies Virus, Human Immunodeficiency Virus 1, Porcine Parvovirus and Hepatitis A Virus) was evaluated. Each validation experiment consisted of spiking the solution at 37 degrees C with virus, heating to 74 +/- 1 degrees C over a period of 30 minutes, holding at 74 +/- 1 degrees C for 90 minutes and cooling from 74 +/- 1 degrees C to less than 10 degrees C over a period of 30 minutes. Duplicate experiments were performed with each of the viruses with the exception of Human Immunodeficiency Virus 1, for which three experiments were performed. In each experiment samples were removed before, during, and after heating for the purpose of determining virus titer and evaluating key process parameters. The results obtained from these experiments confirmed that the key process parameters in these experiments using the scaled down test system reproduced those of the large scale manufacturing process. The results of the virus assays showed at least a 7 log reduction was accomplished by the heat treatment for each of the viruses tested.

Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some PhysiologyBlut ist ein ganz besondrer Saft

“…Terminal sterilization of Hb products is usually achieved by filtration. Some cross-linked materials, being more stable, have been pasteurized. − Complete removal of endotoxins from recombinant Hb produced in pathogenic bacteria is also a challenge …”

Transport and Peripheral Bioactivities of Nitrogen Oxides Carried by Red Blood Cell Hemoglobin: Role in Oxygen Delivery

“…Hb S-nitrosylation (by the mechanisms outlined in reactions [5][6][7][8] is competitive with the dioxygenation reaction (reaction 3) and accounts for a physiological concentration of 10 -7 -10 -5.5 M SNO-Hb in human blood (87,119,123,147,164). Although the preponderant role of O 2 (vs. other electron acceptors) in reaction 5 is still under debate (62,109), crystallographic studies have unequivocally demonstrated both the existence of SNO-Hb and the allosteric control of its generation (22,23).…”

“…Allostery can be further manipulated to align the cell-free Hb's O 2 affinity (which is higher than that of intra-RBC Hb) to physiological values compatible with its clinical use (125,155). Furthermore, various conjugates and cross-linked tetrameric Hb derivatives potently prevent dimerassociated toxicity of cell-free Hb (1) and may prolong Hb shelf-life (6). O 2 affinity is augmented in cell-free Hb not only secondary to dimer formation but also because of the loss of erythrocytic allosteric regulators, such as 2,3-DPG; this difference is important in investigational comparison of cell-free and erythrocytic Hb-NO biology as well as in blood substitute development.…”

αα–Crosslinked Hemoglobin: Was Failure Predicted by Preclinical Testing?

“…General characteristics of the product were published in descriptions of the process, both by Baxter [33, 34, 37, 44, 53]and LAIR [30, 31, 32, 45, 54]. …”

“…The reaction mixture was heated to 60°C for 10 h at pH 7.5, and the less stable unreacted tetramers precipitated out of solution. Although several methods of virus removal and inactivation were evaluated by Baxter [41], this increased stability was exploited to introduce a heat viral inactivation step [42, 43, 44, 45]. The final product was formulated in either Ringer’s acetate, Ringer’s lactate or other physiologic buffer solutions for biological testing.…”