Heat-Stable Hemoglobin via Intramolecular Cross-Linking
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Solution Overview
Problem
Existing hemoglobin-based oxygen carriers face instability issues when removed from red blood cells, leading to renal injury, vasoconstriction, and adverse effects due to dimer formation and protein impurities, and they are not suitable for industrial-scale production.
Innovation Solution
A method involving controlled hypotonic lysis, ultrafiltration, flowthrough column chromatography, and high temperature short time processing to produce a heat-stable, dimer-free, phospholipid-free, and protein impurity-free cross-linked tetrameric hemoglobin, using bis-3,5-dibromosalicyl fumarate for cross-linking and N-acetyl cysteine to prevent met-hemoglobin formation, packaged in an air-tight infusion bag to maintain stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If cross-linking techniques are used to form polymeric hemoglobin to increase circulatory half-life, then the stability of hemoglobin is improved, but met-hemoglobin formation increases and oxygen binding capability decreases
Solution Approach 1:
The invention segments the cross-linking approach by using intramolecular cross-linking within the tetramer to stabilize structure without forming intermolecular polymeric bonds. This selective cross-linking maintains the tetrameric unit as the fundamental stable structure, preventing dimer dissociation while avoiding polymer formation that would reduce oxygen binding capability.
Solution Approach 2:
The invention applies cross-linking locally at specific sites within the hemoglobin tetramer structure rather than allowing random intermolecular cross-linking. This localized intramolecular cross-linking provides structural stability precisely where needed (within the tetramer) without extending bonds between molecules, thereby maintaining oxygen binding sites availability.
2Adaptability or versatility
If hemoglobin is removed from red blood cells to be used as oxygen carrier, then oxygen delivery capability is improved, but renal injury and instability increase due to dimer formation
Solution Approach 1:
The invention performs preliminary intramolecular cross-linking of hemoglobin tetramers before administration to stabilize the structure and prevent subsequent dimer dissociation in circulation. This pre-stabilization ensures that when hemoglobin is released from red blood cells, it maintains tetrameric integrity throughout circulation, preventing renal injury from filtered dimers while preserving oxygen delivery function.
3Object-affected harmful factors
If sulfhydryl reagents are used to prevent vasoconstriction by reacting with hemoglobin sulfhydryl groups, then endothelium-derived relaxing factor binding is prevented, but processing complexity and cost increase
Solution Approach 1:
The invention extracts or removes the need for sulfhydryl reagent treatment by using intramolecular cross-linking to stabilize hemoglobin tetramers. This alternative approach addresses vasoconstriction prevention through structural stabilization rather than chemical modification of sulfhydryl groups, thereby eliminating the complex processing steps and associated costs of sulfhydryl reagent treatment while still preventing harmful vasoconstriction effects.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method produces a stable tetrameric hemoglobin that prevents renal injury and adverse effects, maintains oxygen delivery, and is suitable for industrial-scale production, enhancing oxygenation in tissues and tumors, and improving cancer treatment and organ preservation.
Implementation Method 1
cross-linking techniques to create intramolecular bonds within the tetramer as well as intermolecular bonds between the tetramers to form polymeric hemoglobin
Implementation Method 2
The washed red blood cells are disrupted by a controlled hypotonic lysis for a time sufficient to lyse red blood cells without lysing white blood cells
Implementation Method 3
A first ultrafiltration process is performed using an ultrafiltration filter configured to remove impurities having a higher molecular weight than tetrameric hemoglobin
Implementation Method 4
Flowthrough column chromatography is performed on the second hemoglobin solution to remove protein impurities, dimeric hemoglobin and phospholipids
Implementation Method 5
the cross-linked tetrameric hemoglobin is heat-treated to remove any residual non-cross-linked tetrameric hemoglobin and any non-stabilized hemoglobin, for example the dimeric form of hemoglobin
Implementation Method 6
N-acetyl cysteine to prevent met-hemoglobin formation
Data Source
AI summary
A highly purified and heat stable cross-linked nonpolymeric tetrameric hemoglobin suitable for use in mammals without causing renal injury and vasoconstriction is provided. A high temperature and short time (HTST) heat processing step is performed to remove undesired dimeric form of hemoglobin, uncross-linked tetrameric hemoglobin, and plasma protein impurities effectively. Addition of N-acetyl cysteine after heat treatment and optionally before heat treatment maintains a low level of met-hemoglobin. The heat stable cross-linked tetrameric hemoglobin can improve and prolong oxygenation in normal and hypoxic tissue. In another aspect, the product is used in the treatment of various types of cancer such as leukemia, colorectal cancer, lung cancer, breast cancer, liver cancer, nasopharyngeal carcinoma and esophageal cancer. The inventive hemoglobin can also be used to prevent tumor metastasis and recurrence following surgical tumor excision. Further the inventive tetrameric hemoglobin can be administered to patients prior to chemotherapy and radiation treatment.