Title:  Method for control of functionality during cross-linking of hemoglobins

United States Patent:  6,160,098

Assignee:  Baxter Biotech Technology Sarl (Neuchatel, CH)

Filed:  May 29, 1998

The present invention relates to cross-linked hemoglobins, and particularly to methods of controlling the functionality of such hemoglobins. The controlled functionalities include the P₅₀ and the Hill coefficient. The present invention provides methods for producing a cross-linked hemoglobin with specific final functionalities by regulating the amount of both total hemoglobin and R-state hemoglobin prior to cross-linking, and by modulating cross-linking reaction conditions such as time, temperature, pH and the ratio of cross-linking reagent to hemoglobin.

SUMMARY OF THE INVENTION

This invention relates to methods for preparing crosslinked hemoglobin having target functionalities. In one embodiment of the instant invention, the desired functionality is P₅₀, that is the desired functionality is the oxygen tension for which a hemoglobin solution is half saturated with oxygen. In another embodiment, the desired functionality is the Hill coefficient. The Hill coefficient is a measure of the cooperativity of the hemoglobin molecule.

The method by which the P₅₀ is controlled is accomplished by:

(a) determining initial concentrations of R-state hemoglobin and total hemoglobin in an untreated hemoglobin solution;

(b) comparing the initial concentrations of R-state hemoglobin and total hemoglobin to target concentrations of R-state hemoglobin and total hemoglobin;

(c) if necessary, adjusting the initial concentrations of R state hemoglobin and total hemoglobin to the target concentrations of R-state hemoglobin and total hemoglobin to obtain an adjusted hemoglobin solution; and

(d) chemically treating the untreated hemoglobin solution or the adjusted hemoglobin solutions to obtain crosslinked hemoglobin having a target functionality.

The method by which cooperativity (as measured by the Hill coefficient) is controlled is by modulating at least one "Hill coefficient-affecting paramter" such as the time, temperature, pH, rate of addition of hemoglobin, or molar ratio of the crosslinking reagent to hemoglobin during the crosslinking reaction. The starting hemoglobin is partially, and preferentially fully, deoxygenated prior to crosslinking. The target Hill coefficient is in the range from about 1.0 to 3.0, preferably greater than about 1.7, most preferably about 2.2. The time of treatment is preferably up to about 120 minutes, more preferably less than about 10 minutes, most preferably less than about 1 minute. The pH is preferably in the range from about 6.5 to about 7.5. The molar ratio of crosslinking reagent to hemoglobin is preferentially in the range from about 8:1 to about 12.5:1. The hemoglobin and crosslinkining reagent can be added to the crosslinking reaction simultaneously or sequentially.

The crosslinked hemoglobins prepared by the methods of the present invention are intramolecularly crosslinked, or are both intramolecularly and intermolecularly crosslinked. Methods for crosslinking can be achieved by the use of hetero- or homo-polyfunctional crosslinkers, including, for example, bis-imidoesters, bis-succinimidyl esters and aldehydes. Particularly suitable aldehydes are glycolaldehyde or glutaraldehyde. Such crosslinked hemoglobins an also be pyridoxylated or, preferably, non-pyridoxylated. A further aspect of the present invention is recombinant, mutant hemoglobin that is crosslinked.

Claim 1 of 21 Claims

What is claimed is:

1. A method for preparing a crosslinked hemoglobin solution having a target Hill coefficient comprising:

(a) obtaining a deoxygenated hemoglobin solution;

(b) controlling at least one Hill coefficient-affecting parameter during chemical treatment of said deoxygenated hemoglobin solution, to produce said crosslinked hemoglobin solution having the target Hill coefficient.

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