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-10463733-B1
Publication Date
2019-11-05
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 and antibody ionic liquids that maintain biological activity and heat resistance without the need for refrigeration. It involves cationizing aqueous proteins or antibodies via addition of a positively charged crosslinker in the presence of a coupling reagent, followed by purification, titration with a biologically-compatible counter anionic polymer, repeated dialysis to remove excess anions, lyophilization to remove most water, and controlled heating to form a viscous protein ionic liquid.
The problem addressed is the instability of proteins and antibodies in aqueous environments, where water facilitates hydrolysis, oxidation, and denaturation, leading to short shelf lives and the need for constant refrigeration. Native antibodies in aqueous solutions degrade rapidly at ambient or elevated temperatures and have limited storage life even under refrigeration. Water-free protein preparations are highly desirable to improve stability, eliminate refrigeration dependence, and increase shelf life, especially in environments lacking refrigeration infrastructure.
The invention overcomes limitations by chemically modifying the proteins or antibodies to transform their net charge, enabling formation of stable ionic liquids by pairing cationized antibodies with biologically-compatible anions. This chemical modification carefully maintains biological recognition and antigen-binding specificity, preserving picomolar dissociation constants. The resultant water-free protein ionic liquids are stable at room temperature and resistant to extreme heat (above 100° C.), exhibit long shelf lives without refrigeration, and are biologically compatible and non-toxic.
Claims Coverage
The patent includes two independent claims that focus on methods for creating water-free ultra-stable antibody ionic liquids through specific chemical and processing steps.
Method to create ultra-stable antibody ionic liquid
A method involving: a) providing aqueous anti-hemoglobin antibodies; b) cationizing antibodies by adding stoichiometric amounts of N,N-dimethyl-1,3-propanediamine (DMPDA) with coupling reagents selected from EDC, SIA, 2-(dimethylamino) ethanethiol, or PMPI; c) purifying by repeated dialysis in water; d) titrating with a biologically-compatible counter anionic polymer (poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether) until the solution attains a negative zeta potential; e) dialyzing to remove excess anionic polymer using at least one dialysis membrane with molecular weight cutoff ~7000 g/mol; f) lyophilizing to remove most water; and g) heating between about 27-50 °C to generate the antibody ionic liquid.
Confirmation of cationization by zeta potential measurement
Measuring a positive zeta potential value between about 0 and +5 mV to confirm effective cationization of aqueous antibodies after processing.
Testing temperature resistance and antigen recognition
Heating the protein ionic liquid at about 100° C. for approximately 2 hours and testing antibody ionic liquid recognition of a corresponding antigen, including by dot blot assay on nitrocellulose membrane.
Use of dialysis membranes with specified molecular weight cutoff
Performing dialysis of cationized antibody/anion pairs to remove excess anionic polymer using dialysis membranes with molecular weight cutoffs ranging from about 6000 to 15,000 g/mol to purify protein ionic liquid precursors.
The independent claims describe a detailed multi-step method for synthesizing ultra-stable protein/antibody ionic liquids with controlled cationization, counter anion pairing, purification, lyophilization, and heat processing, confirming stability and biological function at elevated temperatures.
Stated Advantages
Produce ultra-stable, heat-resistant, biologically active protein ionic liquids that do not require refrigeration.
Extend shelf lives beyond traditional aqueous antibody solutions, enabling ambient temperature storage for greater than about five years.
Enable refrigeration-free storage and handling, reducing cost and logistical burden associated with cold supply chains.
Maintain high antigen recognition, specificity, and binding affinity comparable to native antibodies, even after heating to elevated temperatures.
Provide thermostability to temperatures greater than 100° C., preserving biological activity.
Allow easy reconstitution into aqueous or biological buffers for therapeutic use.
Reduce sensitivity to hydrolysis, oxidation, and degradation associated with aqueous biological materials.
Documented Applications
Blood typing via conjugation of IR active dyes to antibodies to enable detection with night vision goggles.
Lateral flow assays and enzyme-linked immunosorbent assays (ELISA) for diagnostic applications.
Anti-venom and anti-toxin therapeutic formulations.
Immunotherapy and vaccine formulations.
Anti-viral agents and detection of chemical, biological, nuclear, environmental, and radioactive agents.
Stabilization of various antibody isotypes (IgG, IgY, IgM, IgE) and biologically-important proteins, including insulin.
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