A METHOD FOR STABILIZING BIOACTIVE MOLECULES INTRODUCTION The present invention relates to a method for preparing stabilized bioactive molecules, in dehydrated form. The method allows for the preparation of bioactive molecules, such as proteins and most particularly antibodies, that may be stored under unfavorable environmental conditions, such as temperature fluctuations and high temperatures.

BACKGROUND OF THE INVENTION It is known in the diagnostic field that proteins, such as monoclonal antibodies, polyclonal antibodies, antigens, ligands and enzymes, are key reagents for the recognition and quantification of biologically relevant molecules. Other analytical techniques utilize nucleic acids. Such reagents are the basis for analytical procedures and diagnostic kits that are commercially available.

Molecules used in analytical procedures and diagnostic kits can have a shelf life of several months or longer if stored at low temperatures, either in liquid (2°C to 8 °C) or solid forms (-30 to-20°C).

When reagent molecules are subjected to stress due to fluctuating temperatures and/or high temperature conditions, they may rapidly lose their biological activity. Thus, commercial products that contain these molecules must be stored under temperature- stabilized conditions in order to protect their biological activity. Whenever the optimal conditions are not met, for example during shipment and delivery of products, the reagent molecules may be damaged.

To obviate the problem of stability, some technical solutions have been proposed which consist of coupling the bioactive molecules to a solid phase (Kochanowska IE, Rapak A, Szewczuk A, "Effect of pretreatment of wells in polystyrene plates on adsorption of some human serum proteins,"Arch Immunol Ther Exp (Warsz) 1994 42: 135-9 ; Ishikawa E, Hamaguchi Y, Imagawa M, Inada M, Imura H, NakazawaN, Ogawa H, "An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay,"J Immunoassay, 1980 1: 3 385-98; Munoz C, Nieto A, Gaya A, Martinez J, Vives J,"New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA, "J Immunol Methods, 1986 Nov 20 94: 1-2 137-44; Kakabakos SE, Livanlou E, Evangelatos GP, Ithakissios DS,"Immobilization of immunoglobulins onto surface-treated and untreated polystyrene beads for radioimmunoassays, "Clin Chem, 1990 Mar 36 : 3 492-6; Ansari AA, HattikudurNS, Joshi SR, Medeira MA, "ELISA solid phase: stability and binding characteristics,"J Immunol Methods, 1985 Nov 28 84: 1-2 117-24; G. Barone, P. Del Vecchio, D. Fessas, C. Giancola, G. Graziano, P. Pucci, A. Riccio, M. Ruoppolo, "Thermal denaturation of ribonuclease T1. A DSC Study, "J. Thermal Analysis, 38 (1992) 2791-2802.) Conjugation of the molecule to a solid substrate permits the storage of product in a non- hydrated form. This approach depends upon a chemical interaction between the reagent and the solid phase and does not allow the subsequent solubilization of the reagent.

However, proteins coupled to solid phases have a good stability only when stored at low temperatures (2°C to 8°C) and may be rapidly inactivated at higher temperatures.

Another system typically utilized for molecule storage is lyophilization (Vallet G. , "Lyophilization or freeze drying. An ideal method of preservation but also one of scientific search for the phytotherapeutic constituents of"Agaricus campestris"or Paris mushroom, "Presse Med, 1966 Mar 26 74: 16 846; List PH, "Lyophilization, freeze drying, "Mitt Dtsch Pharm Ges Pharm Ges DDR 1967 Feb 37: 2 21-6; Gheorghiu M, "Current theoretical principles of lyophilization, "Microbiol Parazitol Epidemiol (Bucur), 1968 Jan-Feb 13: 1 27-38 ; Rueda MK, Derviz GV, Voronov AA, "Study of hemoglobin in erythrocytes after their prolonged preservation in a frozen state and lyophilization," Probl Gematol Pereliv Krovi, 1970 Nov 15: 11 28-32). This method, based on water sublimation starting from ice, cannot be applied to molecules which have a complex quaternary structure, since the method induces conformational changes that may result in loss of biological activity. An additional problem is that the optimal storage temperature for lyophilized material is generally very low, for example,-20°C for thermolabile. enzymes, which can be difficult to achieve and maintain in certain clinical situations.

Therefore, there is a need to develop a method for preparing stabilized dehydrated, preferably anhydrous molecules, for reagent or therapeutic purposes, that are resistant to degradation due to unfavorable environmental conditions. In particular, proteins having the capability to retain biological activity upon exposure to these conditions would be useful for various applications in analytical and diagnostic analysis.

SUMMARY OF THE INVENTION The present invention provides for a method of preparing stabilized dehydrated, preferably anhydrous, bioactive molecules whereby dehydration is accomplished by evaporation rather than sublimation, thereby avoiding the harsh, potentially deactivating conditions associated with lyophilization. In particular, the bioactive molecule to be stabilized, in an aqueous environment, is combined with a detergent, such as a non-ionic detergent, which, without being bound to any particular theory, forms a micelle that preserves the configuration of the bioactive molecule during the evaporation process.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A-B. (A. ) Determination of circulating CD4 positive lymphocytes by flow cytometry using FITC-labelled anti-CD4 monoclonal antibody in liquid form. The percentage of CD4 positive cells was found to be 32.8 percent. The resolution index was calculated to be 7. 33. (B) Determination of circulating CD4 positive lymphocytes by flow cytometry using FITC-labelled anti-CD4 monoclonal antibody in anhydrous form according to the invention. The percentage of CD4 positive cells was found to be 33.1 percent. The resolution index was found to be 7.30. xpos-xheg Resolution index Q Figure 2. Variation of the resolution index over time as a function of storage temperature of anhydrous, FITC-labelled anti-CD4 monoclonal antibody according to the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a method for preparing a biological molecule capable of retaining biological activity when stored under unfavorable conditions.

The method of the present invention comprises the steps of mixing a bioactive molecule with a detergent to create a mixture and evaporating the water in the mixture under controlled conditions.

Various molecules may be used as the bioactive molecules to be stabilized according to the present invention. They include, but are not limited to, monoclonal or polyclonal antibodies, proteins, enzymes, polypeptides, nucleic acids, polysaccharides and lipids. These bioactive molecules may optionally be conjugated to other molecules, for example fluorochromes, enzymes, colloids, isotopes, chemoluminescent substances, or other molecular ligands or labels known in the art.

Preferably, the detergent has minimal de-activating or denaturing effect on the bioactive molecule to be stabilized. In preferred, non-limiting embodiments of the present invention, the detergent is a non-ionic detergent. Detergents which may be used in the present invention include, but are not limited to N-N-bis- (3D-gluconamdopropyl)- cholamide; octaethylenenglycol dodecyl ether; nonaethylenglycol dodecyl ether; cetyltrimethylammonium bromide; 3- (3-cholamidopropyl)-dimethyl-ammonio-l- propanesulfonate ;- (3-cholamidopropyl)-dimethyl-ammonio-2-hydroxi-1- propanesulfonate ; chenodeoxycholic acid; cholic acid; Decyl-ß-D-glucopyranoside ; Decyl-ß-maltoside ; Deoxicholic acid; Digitonin; dodecyl-p-D-maltoside ; N-dodecyl-N- N-dimethylglycine; bis- (2-Ethylexyl) sodiumsulfosuccinate : polyoxyethylene (10) dodecyl ether, polyethylene glycol lauryl ether; glycocholic acid; glycodeoxycholic acid; heptyl-ß-D-glucopyranoside ; heptyl-ß-D-thioglucopyranoside ; hexyl- (3-D- glucopyranoside; lauryldimethylamine oxide; lauryl sulfate; octanoyl-N- methylglucamide; nonaoyl-N-methylglucamide; decanoyl-N-methylglucamide; nonyl-) 3- D-glucopyranoside; NP-40; Octyl-ß-D-glucopyranoside ; octyl-p-D-maltopyranoside ; octyl- (3-D thiogalactopyranoside; octyl- (3-D-thioglucopyranoside ; taurocholic acid; taurodehydrocholic acid; taurodeoxycholic acid; Triton X-100; Triton X-114 ; TWEEN 20; TWEEN 80; N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate ; N-decyl-N, N- dimethyl-3-ammonio-1-propanesulfonate ; N-dodecyl-N, N-dimethyl-3-ammonio-1- propanesulfonate ; and N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N- hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

The amount of detergent added to the molecule to be stabilized is dependent upon the type of detergent used and molecule to be stabilized. Knowledge of such amounts is within the skill of one in the art. For example, but not by way of limitation, the concentraion of bioactive molecule may be between 0.1 picogram and 100 milligram, preferably between 1 picogram and 1 milligram, per milliliter of solution. Preferably, the bioactive molecule to be stabilized is in an aqueous environment (for example, but not by way of limitation, phosphate buffered saline) and the detergent is present at a concentration above its critical micelle concentration, where the critical micelle concentrations for detergents may be found in standard technical reference materials. A non-limiting list of critical micelle concentrations may be found in P. Mukerjee, KJ.

Mysels, Critical Micelle Concentrations of Aqueous Surfactant Systems, NSRDS-NBS, 36 (1971). This concentration is preferably achieved prior to the evaporation process. In specific non-limiting embodiments of the invention, the detergent is present in an amount which solubilizes the compound to be stabilized. According to preferred non-limiting embodiments of the invention, the concentration of detergent is less than or equal to about 1.0 % weight/volume ("w/v").

In an embodiment of the present invention, Tween 20 is mixed with the bioactive molecule to a concentration preferably, but not by way of limitation, greater than about 0.055 mM, or greater than about 0.06 mM, or greater than about 0.07 mM, or greater than about 0.08 mM. Preferably, Tween 20 is added to a final concentration of between 0.008% to 1.0% w/v, preferably between 0.01 to 0.5% w/v, and more preferably 0 0. 1% w/v. Equivalent amounts of other detergents may be used. Preferably, at least 50 mM of Na+ or an equivalent ion is also present.

After the molecule to be stabilized is mixed with the detergent, it is dried such that water in the mixture evaporates. Evaporation may be performed at temperatures which are preferably lower than the temperature at which the molecule to be stabilized denatures and/or is inactivated. Preferably evaporation is performed at ambient temperatures (10-45°C, preferably 15-25°C) although lower or higher temperatures (for example, 4-50°C) may be used provided that the compound to be stabilized is not significantly inactivated. Evaporation is preferably performed under low atmospheric pressure or a vacuum, but may optionally also be carried out at ambient pressure.

The mixture can be dried using varying volumes, with the volume affecting the amount of time required for evaporation. The size and type of the final container during evaporation may also effect the evaporation.

The evaporation step may be performed by placing the dispensed mixtures into a concentrator instrument. The concentrator instrument may, for example but not by way of limitation, be a centrifuge connected to a vacuum pump and having a cool trap for steam condensation.

The evaporation step can be performed not only using the described apparatus, but also without applying centrifugation.

The amount of time required for evaporation depends on the volume of bioactive molecule/detergent solution, the size of the evaporation vessel, the temperature and pressure, factors which may be controlled using techniques known in the art. The completion of the process, when adequate dehydration has been achieved, can also be determined using standard laboratory techniques.

The evaporation step may be performed in the presence, or in the absence of light.

Where light has an altering effect on the compound to be stabilized, it is desirable to perform the various steps in the absence of light.

Preferably evaporation is continued to dehydrate the compound, more preferably until the compound is essentially anhydrous.

Prior to use, it may optionally be desirable to remove the detergent from the composition resulting after evaporation. Such removal may preferably be performed after storage and relatively immediately prior to use. Removal may be performed using standard techniques, such as dialysis.

Without being bound by any particular theory, it is believed that evaporating the mixture generates a water shield around the molecule that imparts a higher stability to the molecule. If the molecule is a protein, dehydrated, and preferably anhydrous proteins are obtained by the water evaporation step. The present invention is superior to lyophilization, since it avoids the temperature stress created during the water sublimation, which could cause the protein to degrade.

The methods of the present invention allows the storage of the stabilized bioactive molecule at elevated temperatures which must be lower than the denaturation temperature of the protein. In addition, the procedure allows an increase in the product shelf life, which may exceed more than 8 years when the product is correctly stored.

Stabilized bioactive molecules according to the invention may be used for diagnostic or therapeutic purposes, as appropriate. The present invention provides for diagnostic or therapeutic compositions comprising such stabilized bioactive molecules, as well as for diagnostic kits comprising such stabilized molecules.

EXAMPLE 1. The stabilization of murine monoclonal antibody conjugated with fluorescein isothiocyanate (FITC).

A monoclonal antibody specific for the CD4 molecule and conjugated to a fluorochrome (fluorescein-FITC) is used as the starting material to generate the molecule of the invention (clone EDU-2 IgG2a isotype). The CD4 molecule is expressed on a monocytes and a subset of T lymphocytes. The CD4 monoclonal is commonly used to determine the number and the percentage of CD4+ T cells in the blood by flow cytometry.

For the present example, the method of the present invention fulfilled dual goals of preserving (1) the ability of the antibody to bind to its antigen and (2) the activity of the photosensitive fluorochrome.

The antibody is diluted to working dilution (65 pg/ml) in saline phosphate buffer (PBS) 10 mM containing NaCl 150 mM, bovine serum albumin (BSA) 1% weight on volume (w/v), natrium azide NaN3 0.2% w/v, pH 7.2-7. 4. To this solution, the detergent is added (Tween 20) to a final concentration of 0. 1 % w/v. After mixing at room temperature away from direct light exposure, 10 gel of solution is dispensed into polystyrene test tubes having 12 mm internal diameter. The tubes are then placed inside a concentrator instrument which consists in a centrifuge connected with a vacuum pump and a cool trap for steam condensation. The tubes are subjected to a 4 hour dehydration process without direct light exposure due to avoid degradation of the photosensitive fluorochrome. At the end of the step, tubes are removed from the centrifuge and capped.

The binding capacity of the anhydrous monoclonal antibody to CD4 is evaluated for the maintenance of biological activity. The resolution index of the anhydrous monoclonal antibody, which is an indicator of the efficiency of the binding between fluorochrome and protein, was also examined. The results of the comparison are reported in Figure 1 and Table I.

The anhydrous monoclonal antibody was also tested for its ability to preserve binding and fluorescence using an accelerated stability test at various high temperatures.

The anhydrous monoclonal antibody were stored at the following three temperatures: 4°C, 37°C and 45°C. At various time points, aliquots were removed from the three temperature conditions from the incubators and tested with a control reference sample by flow cytometry. From such analysis decay curves based upon the resolution index have been drawn (see Figure 2 of Table II).

Key words : protein storage, protein stabilization, high temperature evaporation, surfactants, detergents, anhydrous, dry form.