Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-12-14
2003-06-17
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S007100, C435S007200, C435S007210, C435S068100, C435S069100, C435S069600, C435S173300, C435S173100, C435S252300, C435S320100, C435S811000, C424S085100, C424S133100, C424S135100, C424S145100, C424S810000, C536S023100, C536S023530, C536S023720, C530S387300, C530S388250, C530S388730, C530S868000, C436S008000, C436S016000, C436S018000, C436S176000, C436S548000, C436S826000
Reexamination Certificate
active
06579688
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
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. One example is enveloped viruses, such as influenza viruses of the Orthomyxovirus family that contain antigens that degrade in solution over time at a broad range of storage temperatures. Those of ordinary skill in the art understand that stabilization in solution of various bacterial antigens, for example, some toxins, is also difficult. To avoid deterioration in an aqueous solution those skilled in the art have used lyophilization and freezing as methods for preserving polypeptides or antigens. Indeed, the widespread use of lyophilization and freezing demonstrates the shortcomings and difficulties of preserving such components in aqueous solutions.
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. For example, U.S. Pat. Nos. 5,955,448, 4,496,537, and PCT filing WO 97/04801 all disclose improvements in lyophilization techniques. 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.
This invention concerns an aqueous stabilizing reagent or diluent that enhances the stability of polypeptides, and other non-proteinaceous compounds in solution. Antigens such as carbohydrates, proteins, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, nucleoproteins, and carbohydrates complexed with proteins, lipids, and other compounds are illustrative examples of the types of antigens that may be stabilized using the current invention. Using novel reagent components, the invention improves the stability of polypeptides and antigens in current commercially available diluents or diluents described in the art. The invention is especially useful for stabilizing antigens and polypeptides used as control reagents in diagnostic assays or other uses that require stable aqueous solutions of such components. The stabilizing diluent of the present invention is useful for stabilizing polypeptides and antigens for storage at about 2°-8° C., room temperature and at temperatures of about 45° C. for extended periods of time.
PCT filing No. WO99/15901 discloses a diluent for the stabilization of antigens, in particular, Hepatitis C Virus (HCV) antigens. The HCV diluent comprises a reducing agent to keep the HCV antigens in a reduced form. The publication reports that the inclusion of a reducing agent in a diluent maintained the immunoreactivity of an HCV antigen for up to seven days. The reported diluent further comprises sodium phosphate, pH 6.5 (or other buffer), EDTA (or other chelator), DTT (or other reducing agent), gelatin (or other protein blocking source), ammonium thiocyanate (or other chaotrope), sodium azide (or other preservative) and SDS (or other detergent). 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.
U.S. Pat. No. 4,956,274 concerns techniques for stabilizing peptide fragments from &bgr;-galactosidase for use in complementation assays. The solution disclosed in U.S. Pat. No. 4,956,274 contained an ionic surfactant or a surfactant derived from a sugar residue to slow degradation of &bgr;-galactosidase peptide fragments. 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. The surfactants are neutralized just prior to the assay by using cyclodextrin. Alternatively, the action of the surfactants was masked with a high concentration of serum. Additional components of the disclosed reagent included a chelating agent, buffer, bacteriocide, magnesium or other ions, reducing agents, solubilizing agents such as solvents like ethylene glycol, and nonionic detergents. As those in the art will appreciate denaturing and renaturing proteins or enzyme fragments may damage some antigenic epitopes and render them inactive.
U.S. Pat. No. 5,459,033 describes a solution useful for preventing virus aggregation. The solution was reported to contain N-lauryl sarcosine or other anionic surfactants. The solution was said to enhance stability based on the supposition that virion particles, particularly, hepatitis and herpes viruses aggregate due to hydrophobic attractions thereby decreasing sensitivity. This diluent was not reported to improve or preserve the antigen's catalytic activity or immunogenicity, but to prevent aggregation. Also, before the solution could be used it reportedly had to be incubated for 15 hours to ten days at 2-35° C. to insure consistent stability, adding a major limitation to the manufacturability of the final product.
U.S. Pat. No. 5,660,978 discloses a method of stabilizing an antigen, particularly a labile protein antigen, especially an enzyme, by incorporating into a concentrated solution of antigen (such as serum), an antigen-specific antibody or portions thereof (particularly Fab) to prevent proteolysis or oxidation. Introduction of serum or non-specific IgG would not be expected to provide the desired protection or specificity of protection for such a diluent. Moreover, stabilization was completed prior to placing the antigens in a diluent. In contrast, the current invention is stabilized by the diluent itself. Additionally, the current invention stabilizes a relatively dilute solution from the beginning, not in stages as the patented invention suggests. 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. The invention is essentially utilizing the antibody portions as a fixative reagent, in place of a chemical fixative and thus, does not truly describe a stabilizing diluent.
Landi, S and Held, H R (Tubercle 59 (1978) 121-133) reported the addition of TWEEN® 20 (Polyoxyethylene Sorbitan Monooleate), detergent into a diluted solution and suggested that tuberculin PPD stability is was enhanced by this addition due to the detergent's anti-adsorptive properties. The tuberculin preparation, made by Connaught Laboratories, LTD., contains tuberculin PPD, 0.3% phenol (reported to act as a preservative), and 0.0005% TWEEN® 80 (Polyoxyethylene Sorbitan Monooleate) in PBS. Phenol is a hazardous material that would be unacceptable in the current invention and is li
Panzarella Laura
Steaffens Jeffrey W.
Biostar Inc.
Cook Lisa
Foley & Lardner
Le Long V.
Warburg Richard J.
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