Ultrastable antibody ionic liquids
Inventors
Slocik, Joseph M • Naik, Rajesh R. • Dennis, Patrick B
Assignees
United States Department of the Air Force
Publication Number
US-11338037-B1
Publication Date
2022-05-24
Expiration Date
2037-02-23
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Abstract
A method for creating a stable protein/antibody ionic liquid, comprising: (a) cationizing aqueous proteins/antibodies by addition of an excess of a positively-charged crosslinker in the presence of a coupling reagent; (b) purifying the cationized proteins/antibodies; (c) titrating the cationized proteins/antibodies with a corresponding biologically-compatible counter anionic polymer to create at least one protein/antibody cation/anion pair in aqueous solution until the cation/anion pair solution becomes negative by zeta potential measurement; (d) repeatedly dialyzing the protein/antibody cation/anion pair in water to remove excess anionic polymer using at least one molecular weight cutoff 7000 dialysis membrane; (e) lyophilizing the protein/antibody cation/anion pair to remove most of the water, forming a lyophilized solid; and (f) heating the lyophilized solid until a protein/antibody ionic liquid is generated. The antibody may be any desired antibody, and the anion may be any biologically-compatible anion.
Core Innovation
The invention provides a method for creating ultra-stable, water-free protein or antibody ionic liquids by chemically modifying aqueous proteins or antibodies into ionic liquids that maintain biological activity without the need for refrigeration. This method involves cationizing the proteins or antibodies by adding a positively charged crosslinker in the presence of a coupling reagent, purifying the cationized proteins, titrating with a biologically-compatible counter anionic polymer until the solution becomes negatively charged by zeta potential measurement, dialyzing to remove excess anion, lyophilizing to form a solid, and heating the solid to generate the ionic liquid.
The problem addressed by the invention arises from the instability of biological materials such as proteins and antibodies in aqueous environments, where water promotes hydrolysis, oxidation, and denaturation leading to short shelf lives and the requirement of refrigeration for storage and transport. Water increases the sensitivity of antibodies to elevated temperatures, destabilizes protein structure, and accelerates degradation processes. The invention aims to overcome these issues by removing most or all of the water from antibody preparations while preserving their antigen recognition, specificity, and binding affinity, resulting in a heat-resistant, biologically active, water-free ionic liquid stable at room temperature and beyond.
This method results in protein or antibody ionic liquids that are viscous, clear liquids resistant to extreme temperatures (greater than 100° C.), possess long shelf lives exceeding five years, and do not require refrigeration. By selectively cationizing acidic groups such as carboxyl, amine, and hydroxyl groups on the antibodies, biological functionality is retained. The ionic liquids can be reconstituted for therapeutic use and exhibit significantly improved stability and handling compared to traditional aqueous antibody solutions.
Claims Coverage
The patent contains one independent claim detailing a method for creating a water-free ultra-stable antibody ionic liquid with several inventive features.
Method for creating a water-free ultra-stable antibody ionic liquid
The method includes providing at least one aqueous anti-hemoglobin antibody; cationizing the antibody by adding stoichiometric amounts of N,N-dimethyl-1,3-propanediamine (DMPDA) with a coupling reagent chosen from EDC, SIA, 2-(dimethylamino) ethanethiol, and PMPI; purifying the cationized antibody by repeated dialysis in water; titrating the cationized antibody with a counter anionic polymer of poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether until the solution becomes slightly negative by zeta potential measurement; dialyzing the antibody cation/anion pair to remove excess anionic polymer using membranes with molecular weight cutoff between about 6000-15,000 g/mol; lyophilizing to remove most water to form a solid; and heating the lyophilized solid at about 27-50° C. until the antibody ionic liquid is formed.
Dialysis with specific molecular weight cutoff membrane
The method includes performing dialysis using at least one membrane with a molecular weight cutoff of about 7000 g/mol to remove excess reagents and salts from the cationized antibody.
Confirmation of cationization by zeta potential measurement
The method comprises confirming successful cationization of the antibody by measuring a positive zeta potential value between about 0 and +5 mV to determine the number of added positive charges.
Thermal stability testing of antibody ionic liquid
The method comprises heating the antibody ionic liquid at about 100° C. for about 2 hours and testing its antibody recognition of the corresponding hemoglobin antigen using a dot blot assay on a nitrocellulose membrane to confirm retained biological activity.
The independent claim recites a multi-step method to produce water-free, ultra-stable antibody ionic liquids by cationizing antibodies, balancing with a compatible anionic polymer, purifying and lyophilizing the product, and heating to generate the stable ionic liquid, including steps to confirm cationization and test thermal stability.
Stated Advantages
The antibody ionic liquids are ultra-stable with shelf lives greater than about five years.
They retain biological activity and antibody recognition even after exposure to temperatures exceeding 100° C.
They do not require refrigeration for storage, handling, or transport, eliminating cold supply chain needs.
The method reduces costs and logistical burdens associated with refrigeration and heavy cooling equipment.
They are biologically compatible and non-toxic.
The ionic liquids can be easily reconstituted into aqueous buffers for therapeutic use.
Documented Applications
Use in antibody-based diagnostics allowing refrigeration-free handling and transport.
Preparation of blood-typing antibodies conjugated with IR active dyes for non-visible light detection with night vision goggles, aiding soldiers and field medics.
Applications in lateral flow assays, enzyme-linked immunosorbent assays (ELISA), anti-venom and anti-toxin therapeutics, immunotherapy, vaccines, anti-virals.
Detection of chemical, biological, nuclear, environmental, and radioactive agents.
Stabilization of other biologically important proteins, including negatively or positively charged proteins such as insulin.
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