Ultra-stable protein ionic liquids

Inventors

Slocik, Joseph M • Naik, Rajesh R. • Dennis, Patrick B

Assignees

United States Department of the Air Force

Abstract

A method comprising: providing aqueous insulin; titrating the aqueous insulin with a mixture of small molecule anions, e.g. D- and L-amino acid esters, small D- and L-peptide pairs, and DL lactate solution, to form an insulin/anion pair solution. Titrating may be performed until the insulin/anion pair solution becomes negative by zeta potential measurement. The insulin/anion pair solution may be dialyzed to remove excess anionic polymer using a membrane sufficient to separate the insulin/anion pairs from excess small molecule anions. The insulin/anion pair solution may be dialyzed or lyophilized to remove all of the water, forming a solid of ultra-stable insulin. The positive electrostatic charge of the aqueous insulin may be confirmed by measuring a positive zeta potential value.

Core Innovation

The invention relates to ultra-stable, heat-resistant, biologically active, water-free protein ionic liquids that do not require refrigeration. The method involves providing aqueous insulin and titrating the aqueous insulin with a mixture of small molecule anions, such as D- and L-amino acid esters, small peptide pairs, and DL lactate solution, forming an insulin/anion pair solution. Titration is continued until the insulin/anion pair solution becomes negative as measured by zeta potential. Dialysis can remove excess anionic polymer, and lyophilization removes water, resulting in a solid ultra-stable insulin. The positive electrostatic charge of aqueous insulin is confirmed by measuring positive zeta potential.

The problem addressed is that most biological materials, like proteins and antibodies, normally require aqueous environments and refrigeration to maintain stability and biological function due to water's dual role of stabilizing but also promoting degradation processes such as hydrolysis and oxidation. Water increases susceptibility to elevated temperatures and leads to denaturation and short shelf lives. Current biological materials require constant refrigeration, but water presence results in destabilization and short half-lives without such refrigeration. Many regions lack electricity for refrigeration, limiting storage and use.

This invention overcomes these problems by removing most or all water (at least 95%), transforming proteins and antibodies into ionic liquids through cationization and subsequent pairing with biologically compatible anions. This maintains antigen recognition, specificity, and binding affinity similar to native antibodies. The water-free protein liquids combine stability, resistance to extreme temperatures (over 100 °C), and significantly extended shelf lives without refrigeration. The methods include cationizing proteins using positive crosslinkers and coupling agents, titrating with biologically compatible counter anions, dialyzing to remove excess reagents, lyophilizing to remove water, and heating to generate viscous protein ionic liquids.

Claims Coverage

The patent contains one independent claim detailing a method for preparing ultra-stable insulin ionic liquids.

The claims encompass methods to produce ultra-stable insulin ionic liquids involving controlled titration with biologically compatible anions, purification by dialysis, water removal by lyophilization, and heating to form viscous stable ionic liquids, with validation by zeta potential measurements.

Stated Advantages

Documented Applications