Stabilizing diluent for polypeptides and antigens

Abstract

Compositions for stabilizing polypeptides or antigens are described. These compositions are useful for stabilizing polypeptides or antigens stored in aqueous formulations. Such formulations can be used for various analytical or other methods.

Description

[0001] This application is related to U.S. provisional patent application No. 60 / 170,850, entitled "STABILIZING DILUENT FOR POLYPEPTIDES AND ANTIGENS," from which priority is claimed, and which is hereby incorporated by reference in its entirety, including all claims, figures, and tables.[0002] The present invention relates to aqueous compositions useful for stabilizing polypeptides and antigens. The stabilized polypeptides and antigens are useful in analytic methods such as antigen-specific detection, as well as other pharmaceutical uses where the stabilization of such components in an aqueous solution is desirable.[0003] The following is a discussion of literature potentially relevant to the invention disclosed herein. However, none of the references discussed herein is admitted to be prior art.[0004] Stabilizing polypeptides and antigens in aqueous solutions is often difficult. For example, storage of such solutions at room temperature for prolonged periods results in deteriorati...

AI Technical Summary

Benefits of technology

[0014] The disclosed reagent surpasses previous formulations for polypeptide and antigen stability at both low and elevated temperatures. Polypeptides and antigens can be stored in the reagent in soluble form for extended periods of time at a variety of temperatures from about 0.5.degree. C. to more than about 50.degree. C., preferably from about 2-8.degree. C., about room temperature (typically from about 23.degree. C. to about 28.degree. C., with 25.degree. C. being particularly preferred) and about 42.degree. C. to about 43.degree. C., especially about 45.degree. C. Stability at 45.degree. C. is predictive of the reagent's ability to provide long-term antigen stability. Additionally, the disclosed reagent has the advantage of being a single aqueous solution for the purpose of stabilizing polypeptides and antigens.

[0056] This experiment tested the ability of the cell culture media (EMEM, BioWhittaker Cat. #12 136Q) to enhance stability of the antigens as a component of the inventive stabilizing diluent. Four test solutions were made: (A) 75% inventive diluent, 12.5% EMEM and 12.5% Stabilcoat buffer, (B) solution A, where the EMEM additionally contains 2% FCS, 1% L-Glutamine, and 0.5% PenStrep / Fungizone (an antimicrobial solution, BioWhittaker Cat. #17-745H), (C) 75% inventive diluent and 25% Stabilcoat diluent, and (D) inventive diluent only. A 1:10 dilution of inactivated influenza B stock suspension was made in each of the four diluents by first making a 1:4 dilution and subsequently a 1:2.5 dilution with the respective test diluent. Each solution was tested at Day 3 at 4.degree. C. and Day 3 at 45.degree. C. Solution A showed the best stability at 3 days at 45.degree. C., followed by solution B, D and C. The results indicate that incorporating cell culture media / Stabilcoat.RTM. into the diluent under increasingly dilute antigen conditions enhances the stability of the antigen (compare stability in solution A to that of solution D). Utilizing a higher concentration of Stabilcoat.RTM. buffer as the only other constituent (solution C) proved deleterious to the antigen's stability (relative to solution A).

Problems solved by technology

However, none of the references discussed herein is admitted to be prior art.

Stabilizing polypeptides and antigens in aqueous solutions is often difficult.

For example, storage of such solutions at room temperature for prolonged periods results in deterioration of polypeptides or antigens contained in the aqueous solution.

In particular, antigens of bacteria, viruses, and other microorganisms have been documented to be unstable when stored in an aqueous medium.

Those of ordinary skill in the art understand that stabilization in solution of various bacterial antigens, for example, some toxins, is also difficult.

Numerous publications in the art disclose means of increasing the stability of a lyophilized reagent, and the difficulties inherent even within this state of the art technology.

Lyophilization of protein containing solutions, however, imposes major costs and inconvenience on the manufacturer and the end user, as well as introducing an increased risk for reconstitution errors and contamination.

Additionally, freezing of a solution requires special equipment and ultimately can lead to protein degradation when repeated cycling occurs.

For commercial use, deterioration of polypeptides and antigens in aqueous solutions is costly because such solutions require replacement after only a short storage life.

However, the inclusion of a reducing agent in the diluent may be ineffective in or deleterious to the stability of many antigens from other microorganisms.

However, the surfactants also denatured the enzyme fragments, and thus had to be removed or neutralized to enable the enzymatic fragments to return to their correct conformation and regain enzymatic activity, indicating that the solution did not stabilize the native form of the protein.

As those in the art will appreciate denaturing and renaturing proteins or enzyme fragments may damage some antigenic epitopes and render them inactive.

Also, before the solution could be used it reportedly had to be incubated for 15 hours to ten days at 2-35.degree. C. to insure consistent stability, adding a major limitation to the manufacturability of the final product.

Introduction of serum or non-specific IgG would not be expected to provide the desired protection or specificity of protection for such a diluent.

The use of antibodies to structurally stabilize an antigen would be problematic especially if one or more of the antigenic sites are the target of an immunological assay.

Additionally, one skilled in the art would have difficulty consistently producing a diluent such as the one disclosed in the '978 patent, which conformationally preserves the antigen, but does not inhibit specific enzyme activity.

Phenol is a hazardous material that would be unacceptable in the current invention and is likely to denature some proteins.

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