FIELD OF THE INVENTION

The present invention relates generally to ultra-stable, water-free biological materials and, more particularly, to ultra-stable, heat-resistant, biologically active, water-free protein ionic liquids that do not require refrigeration.

BACKGROUND OF THE INVENTION

Most biological materials (i.e. proteins and antibodies) thrive in aqueous environments and physiological conditions (neutral pH—between 6-8, ambient temperatures 25-37° C.) in order to perform their biological function. Water is used for stabilizing some biomolecular structures through hydrogen bonding, providing proton donors/acceptors, regulating binding interactions, and controlling molecular dynamics. Conversely, water is also detrimental to biomolecular structure and function by increasing the rate of hydrolysis and oxidation, destabilizing the protein structure, and increasing the susceptibility/sensitivity to elevated temperatures. In total, this results in denaturation, proteolytic degradation, decomposition, and short shelf-lives of these materials.

In order to counteract the effects of water and limit decomposition, current biomolecules, e.g. proteins and antibodies, may require constant refrigeration during storage, handling, and transport in order to preserve their structure, functionality, and biological activity. Generally, antibodies in water may be stable for up to one month when stored at about 4° C. and up to one year when stored in 25% glycerol at −20° C. However, the presence of water in a biological solution will typically result in hydrolysis, even if the temperature is reduced or the solution is frozen. Water promotes hydrogen bonding, intramolecular interactions, stabilizes the antibody structure, facilitates mass transport and diffusion of products, and regulates binding interactions. Water also increases the sensitivity of the antibodies to elevated temperatures, destabilizes protein structures, increases hydrolysis and oxidation rates, reduces shelf lives, and promotes unfolding/denaturation. Consequently, the exclusion of water from antibody preparations is highly appealing and offers a means towards reducing protein degradation, increasing stability, enabling refrigeration-free storage and handling, and significantly increasing shelf-lives. In addition, even if freezing or refrigeration are acceptable alternatives, many places around the world have no available electricity to power refrigeration equipment. The half-life of unrefrigerated antibodies may be as short as 2 days.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing problems and other shortcomings, drawbacks, and challenges of making stable biologically-active materials, such as proteins and antibodies. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. To the contrary, this invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the present invention.

Based on the numerous drawbacks associated with water in antibody solutions, see above, the aim of this invention, in one embodiment, is the removal of most or all water, i.e. at least 95% water removed or at least 99% water removed, without disrupting the protein/antibody structure and intramolecular interactions/functions. The proteins and/or antibodies are cationized, anionized, and chemically modified into an ionic liquid, but when antibodies are used the modified antibodies maintain high antigen recognition, specificity, and binding affinity, e.g., the modified antibodies maintain picomolar (pM) dissociation constants (KD) about equal to those of native, unmodified antibodies. With regard to binding affinity, this means that the antigens bind strongly to the modified antibodies.

“Water-free” (as defined herein) protein liquids feature the simplicity of traditional inorganic ionic liquids (facile synthesis, ability to tune properties through choice of cation and anion pair, and stability), but display the complexity and functionality of highly active proteins, e.g. antibodies. Because the protein liquids have most or all of the water removed, they are stable liquids, may be resistant to extreme temperatures (>100° C.), able to maintain biological recognition activity, and exhibit much longer shelf-lives without the need for refrigeration.

According to one embodiment of the present invention a method for creating a stable protein ionic liquid, comprises: (a) cationizing aqueous proteins by addition of an excess of a positively-charge crosslinker in the presence of a coupling agent; (b) purifying the cationized proteins; (c) titrating the cationized proteins with a corresponding biologically-compatible 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; (d) dialyzing the at least one protein cation/anion pair in water at least once to remove excess anionic polymer using at least one molecular weight cutoff 7000 dialysis membrane; (e) lyophilizing the at least one protein cation/anion pair to remove most of the water, forming a lyophilized solid; and (f) heating the lyophilized solid until a protein ionic liquid is generated. In cationizing the aqueous proteins, a minimum zeta potential value of +5 mV is desired for cationization. In titrating the cationized antibodies, the negative zeta potential is meant below 0 mV to about −1 mV by zeta potential. A negative zeta potential of the titrated cationized antibodies ensures that there is a minor excess of anion but that the positive charges are equally balanced. Heating of the lyophilized solid may be done on a hotplate, in a temperature controlled water bath, or an oven at about 27-50° C., for example. This provides the advantage of producing stable, heat-resistant, biologically active protein ionic liquids that do not require refrigeration. In one embodiment of the present invention, the protein ionic liquid is a viscous, clear liquid. Antibodies may include but are not limited to IgG, IgY, IgM, and other proteins or negatively-charged molecules may also be rendered stable according to the teachings herein.

According to a first variation, the method for creating a stable protein ionic liquid further comprises purifying the cationized proteins from excess coupling reagents by dialysis in water. This provides the advantage of obtaining a pure protein sample composed of only proteins modified with positive charges.

According to another variation, the method for creating a stable protein ionic liquid further comprises cationizing aqueous proteins by addition of an excess of or a stoichiometric amount of N,N-dimethyl-1,3-propanediamine crosslinker in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reagent.

According to a further variation, the method for creating a stable protein ionic liquid further comprises cationizing aqueous proteins by addition of an excess of or a stoichiometric amount of 2-(dimethylamino) ethanethiol crosslinker in the presence of succinimidyl iodoacetate (SIA) coupling agent.

According to a further variation, the method for creating a stable protein ionic liquid further comprises cationizing aqueous proteins by addition of an excess of or a stoichiometric amount of 2-(dimethylamino) ethanethiol crosslinker in the presence of N-(p-maleimidophenyl) isocyanate (PMPI) coupling agent.

According to another variation, the method for creating a stable protein ionic liquid further comprises performing the dialysis with at least one membrane with a molecular weight cutoff (MWCO) of about 7000 g/mol. According to a further variation, the method for creating a stable protein ionic liquid further comprises performing the dialysis with at least one membrane with a molecular weight cutoff of between about 6000-15,000 g/mol. In one embodiment this membrane may remove a plurality of contaminants and excess reagents from the modified proteins that are below a molecular weight, e.g. 7000 g/mol. A molecular weight of at least about 7000 g/mol typically ensures that all coupling reagents, positively-charged cross-linker, and buffer salts are separated from cationized proteins. About 7000 g/mol may be the lower limits for this dialysis, however, the membrane could be as large as 15,000 g/mol, but at the risk of losing proteins through the larger membrane.

According to a further variation, the method for creating a water-free ultra-stable protein ionic liquid further comprises confirming the cationizing of the aqueous proteins by measuring a positive zeta potential value. The zeta potential may be between about 0 mV and +5 mV. This provides the advantage of determining the number of positive charges added to the protein.

According to another variation, the method for creating a stable protein ionic liquid further comprises titrating the cationized proteins with the corresponding biologically-compatible counter anionic polymer of poly(ethylene glycol) 4-nonylphenyl 3-suopropyl ether, i.e. C9H19C6H4—(OCH2CH2)20O(CH2)3SO3. In other embodiments the counter anion polymer may be biologically-derived DL-Lactate, biologically-derived linolenic acid, phospholipids, fatty acids, the conjugate base form of amino acids (i.e. deprotonated and negatively charged), any biologically-derived singly-charged anion with low melting points (e.g. between about 5-30° C.). This provides the advantage of balancing the positive charges on the protein with negative charges of the anion to form the ionic salt form of the protein.

According to a further variation, the method for creating a stable protein ionic liquid further comprises heating the lyophilized solid to about 50° C. to generate the protein ionic liquid. This provides the advantage of melting the protein ionic salt to form a viscous water-free liquid without deactivating the protein.

According to another embodiment of the invention, the protein may be an antibody.

According to another variation, the method for creating a stable protein ionic liquid further comprises heating the protein ionic liquid at about 100° C. for about 2 hours; and testing the protein ionic liquid for antibody recognition of a corresponding antigen, when the protein is an antibody. In one embodiment, the testing may be done using a dot blot assay on a nitrocellulose membrane. In a further embodiment, the heating may be between about 75° C. and about 150° C. and/or may be between 1 and 3 hours. This provides the advantage of evaluating the temperature stability of the protein/antibody ionic liquid at extreme temperatures by directly measuring binding activity of the antibody for an antigen.

According to a further variation, the protein is an anti-hemoglobin antibody, polyclonal anti-horse spleen ferritin antibodies, monoclonal Anti-Flag antibodies, monoclonal Anti-HRP2 toPlasmodium falciparum, polyclonal Anti-neuropeptide Y, polyclonal Anti-human troponin, and all antibody isotypes, e.g. IgY, IgG, IgM, IgE, etc.

In addition, a dye, such as an IR active dye, may be combined with blood-typing antibody solutions via conjugation of an amine reactive dye, e.g., Anti-A (IgM antibody) ionic liquid, such that blood typing may be accomplished without visible light using night vision goggles to determine blood type via the hemagluttination of red blood cells, a tremendous boon to soldiers and field medics in hazardous regions. Other variations may be useful for lateral flow assays, enzyme-linked immunosorbent assays (ELISA), anti-venom/anti-toxin therapeutics, immunotherapy, vaccines, anti-virals, detection of chemical, biological, nuclear, environmental and radioactive agents, and may be applied to other biologically-important proteins whether negatively or positively charged, e.g., insulin.

According to a second embodiment of the invention, a method comprises the steps of: providing aqueous insulin; and titrating the aqueous insulin with a mixture of small molecule anions (i.e. DL lactate or polymer anions) to form at least one enzyme/anion pair in aqueous solution.

According to a first variation of the second embodiment, the small molecule anions may be one or more of D- and L-amino acid esters, small D- and L-peptide pairs, and DL lactate solution, i.e. no more than 60 wt % DL lactate solution. It may be desirable for any anions used to be non-toxic and biocompatible. The anions may be racemic mixtures. The titration step is performed until the insulin/anion pair solution becomes negative by zeta potential measurement. Currently, shipping and storage of insulin requires that the mixture is kept cool, but not frozen. Converting the insulin mixture into an ionic liquid would eliminate the need for this.

According to a further variation of the second embodiment, the method may further comprise dialyzing the at least one insulin/anion pair to remove excess anionic polymer using a dialysis membrane having a molecular weight cutoff suitable for separating insulin/anion pairs from the solution, e.g. between 2000 and 3500 g/mol. The molecular weight of insulin is 5700 g/mol, so an acceptable cutoff range of dialysis membrane would be about 2000 to 3500 g/mol. The dialysis membrane may be selected to remove to selectively separate the desired components. The dialysis step is optional but may be considered important if high purity, i.e. for medical or therapeutic human or animal use, is desired. A dialysis (or purification) step may be performed before and/or after titration to purify the components at each stage of the method. Where dialysis is performed before titration, it may be helpful to distinguish the dialysis step as ‘purifying’ the insulin. Regardless of whether it is called dialysis or purification, this provides the advantage of obtaining a pure insulin samples composed of only insulin and/or insulin/anion pairs. The particular molecular weight target may be selected according to the desired results and the particular insulin or insulin/anion pairs being processed.

According to another variation of the second embodiment, the method may further comprise lyophilizing the insulin/anion pair solution to remove most or all of the water, forming a lyophilized solid of ultra-stable insulin. The amount of water remaining may be determined by thermal gravimetric analysis. The lyophilized insulin/anion pairs are shelf-stable and do not require refrigeration. Removing “most of the water”, “all of the water”, or “water-free” requires removal of at least 95% of the water, and in some embodiments more than 99% of the water. Lyophilization may time some time, depending on how much water is present. A 1 mL sample, for example, may take 10-12 hours, for example.

According to a further variation of the second embodiment, the method further comprises confirming the positive (electrostatic) charge of the aqueous insulin by measuring a positive zeta potential value. A minimum zeta potential value between 0 and about +5 mV may be desired for further processing, i.e. titration with anions. This step is optional, but may be useful simply to confirm that the insulin is suitable for further processing.

According to another variation of the second embodiment, when titrating the aqueous insulin, the negative zeta potential desired is below 0 mV to about −1 mV by zeta potential. A negative zeta potential of the titrated insulin/anion pairs ensures that there is a minor excess of anion but that the positive charges are equally balanced.

According to a further variation of the second embodiment, the method may further comprise heating the lyophilized solid at about 40-90° C. until an insulin ionic liquid is generated. This may take approximately 20-30 minutes, or longer, of heating. In one embodiment of the present invention, the insulin ionic liquid is a viscous, clear liquid.

According to another variation of the second embodiment, the method may further comprise heating the water-free ultra-stable insulin ionic liquid up to about 50° C. for at least 20 minutes.

According to a further variation of the second embodiment, the insulin ionic liquid may be tested for functionality and stability by using insulin responsive cells for chemiluminescence or amount of phosphorylation. Stability can also be determined by measuring structure of insulin by circular dichroism, infrared spectroscopy, or a thermal shift assay. These optional steps relates to the determination of the thermal limits appropriate for storage, i.e. long-term unrefrigerated storage, of the stabilized insulin. Although 50° C. is an upper limit provided herein, different thermal limits, e.g. 30° C., 100° C., 200° C., or temperatures anywhere between may be chosen, depending on the required efficacy of the stabilized insulin and the relative fragility of the insulin. Heating of the lyophilized solid may be done on a hotplate, in a temperature controlled water bath, or an oven at about 27-50° C., for example. The optional heating step demonstrates that a stable, heat-resistant insulin ionic liquid is formed.

According to a third embodiment of the invention, a water-free ultra-stable insulin ionic liquid comprises an insulin/anion pair. Such insulin ionic liquids may be made according to the disclosed method.

According to a variation of the third embodiment of the invention, the water-free ultra-stable insulin ionic liquid may further comprise an anionic polymer, e.g. small molecule anions, e.g. D- and L-amino acid esters, small D- and L-peptide pairs, and a 50 wt % DL lactate solution.

Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

