Ultra-stable protein ionic liquids
Inventors
Slocik, Joseph M • Naik, Rajesh R. • Dennis, Patrick B
Assignees
United States Department of the Air Force
Publication Number
US-11274163-B1
Publication Date
2022-03-15
Expiration Date
2037-02-23
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Abstract
A method for creating an antibody ionic liquid, comprising: providing aqueous anti-A, anti-B, or anti-Rh antibodies; cationizing the aqueous antibodies by the addition of stoichiometric amounts of an excess of a positively-charged crosslinker in the presence of a coupling reagent; titrating the cationized antibodies with a counter anionic polymer to create at least one antibody cation/anion pair in aqueous solution until the antibody cation/anion pair solution becomes negative by zeta potential measurement. The method may further comprise lyophilizing the antibody cation/anion pair solution to remove all of the water, forming a lyophilized solid; and heating the lyophilized solid to generate a protein ionic liquid. Cationized antibodies may be purified of excess coupling reagents by dialysis in water by a membrane, and the antibody cation/anion solution may be dialyzed to remove excess anionic polymer with a similar membrane.
Core Innovation
The invention provides a method for creating ultra-stable, water-free biological materials, particularly protein ionic liquids composed of antibodies that remain biologically active and heat-resistant without requiring refrigeration. The method involves cationizing aqueous antibodies using stoichiometric amounts of a positively-charged crosslinker in the presence of a coupling reagent, then titrating the cationized antibodies with a counter anionic polymer until the solution reaches a negative zeta potential, indicating charge balance. Subsequently, the antibody cation/anion pair solution is lyophilized to remove water and heated to generate a protein ionic liquid.
The problem being solved addresses the instability and short shelf-life of proteins and antibodies in aqueous environments, where water promotes degradation through hydrolysis, oxidation, denaturation, and temperature sensitivity, necessitating refrigeration and cold supply chains. This limits storage, transport, and use, especially in locations without reliable refrigeration. The invention's approach removes most or all water (at least 95%) while maintaining antibody structure and function, thus producing a water-free ionic liquid that is stable at elevated temperatures (above 100° C.), preserves antigen recognition and binding affinity, and exhibits potentially extended shelf-lives without refrigeration.
Claims Coverage
The claims include two independent claims: one directed to a method for creating a stable antibody ionic liquid, and another reciting a stable antibody ionic liquid composition. These claims cover the inventive features related to the preparation and composition of the antibody ionic liquids.
Method for creating a stable antibody ionic liquid
This method comprises providing aqueous blood typing antibodies (anti-A, anti-B, or anti-Rh), cationizing them by addition of stoichiometric amounts of positively-charged crosslinkers (including N,N-dimethyl-1,3-propanediamine with EDC, succinimidyl iodoacetate with 2-(dimethylamino) ethanethiol, and/or N-(p-maleimidophenyl) isocyanate with 2-(dimethylamino) ethanethiol), and titrating the cationized antibodies with a counter anionic polymer (exemplified by poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether) to form cation/anion pairs until a negative zeta potential is reached.
Removal of water by lyophilization to form a lyophilized solid
After formation of the antibody cation/anion pair solution, it is lyophilized to remove essentially all water, forming a lyophilized solid which is the precursor to generating the ionic liquid.
Generation of antibody ionic liquid by heating
The lyophilized solid is heated (e.g., from about 27 to 50° C.) for a time sufficient to generate a viscous, clear antibody ionic liquid that retains biological activity and stability.
Purification steps for cationized antibodies and antibody cation/anion pairs
Dialysis is used to purify cationized antibodies from excess coupling reagents and to remove excess anionic polymer from antibody cation/anion pair solutions, employing membranes with molecular weight cut-offs around 7000 g/mol or within 6000–15,000 g/mol range.
Confirmation of cationization by zeta potential measurement
The successful cationization of aqueous antibodies is confirmed by measuring a positive zeta potential value, typically between 0 and +5 mV.
Stable antibody ionic liquid composition comprising specific antibody cation/anion pairs
The composition includes anti-hemoglobin antibody cation/anion pairs with antibodies selected from blood typing anti-A, anti-B, and anti-Rh antibodies, combined with anionic polymers such as poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether, DL-lactate, linolenate, phospholipids, fatty acids, conjugate base forms of amino acids, biologically-derived singly charged anions, or small DL-peptide pairs. Crosslinkers and coupling reagents are selected from specified chemical groups including N,N-dimethyl-1,3-propanediamine and EDC or succinimidyl iodoacetate.
The claims collectively cover methods for preparing stable antibody ionic liquids by cationization and anion pairing, removal of water, and heating to form heat-resistant and biologically active ionic liquids, as well as the compositions of such stable antibody ionic liquids comprising specific antibodies, crosslinkers, coupling reagents, and biocompatible anionic polymers.
Stated Advantages
The protein ionic liquids are ultra-stable and resistant to extreme temperatures exceeding 100° C.
They maintain high biological activity, including antigen recognition, specificity, and binding affinity equivalent to native antibodies.
These materials have long shelf-lives (extrapolated to greater than five years) without the need for refrigeration or adherence to cold supply chains.
They reduce costs and logistical burdens associated with refrigeration, including the elimination of heavy refrigeration equipment weight.
They are non-toxic, biologically compatible, and can be easily reconstituted into aqueous or biological buffers for therapeutic use.
Documented Applications
Blood typing using anti-A, anti-B, and anti-Rh antibody ionic liquids that retain hemagglutination capability even after heating to 100° C.
Lateral flow assays and enzyme-linked immunosorbent assays (ELISA).
Anti-venom and anti-toxin therapeutics.
Immunotherapy and vaccine formulations.
Antiviral agents.
Detection of chemical, biological, nuclear, environmental, and radioactive agents.
Application to other biologically-important proteins, including insulin.
Use with dye-labeled antibody ionic liquids enabling blood typing via infrared detection with night vision equipment for battlefield or field use without visible light.
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