OA12935A - A method of stabilizing bioactive molecules. - Google Patents

Abstract

A method for preparing stabilized molecules, in dehydrated form, is provided. The method allows for the preparation of molecules, such as proteins and most particularly antibodies, that may be stored under unfavorable environmental conditions, such as temperature fluctuations and high temperatures.

Description

1 012935

1 A METHOD FOR STABILIZING BIOACTIVE MOLECULES

INTRODUCTION

The présent invention relates to a method for preparing stabilized bioactive5 molécules, in dehydrated form. The method allows for the préparation of bioactive molécules, such as proteins and most particularly antibodies, that may be stored underunfavorable environmental conditions, such as température fluctuations and hightempératures.

10 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 récognitionand quantification of biologically relevant molécules. Other analytical techniques utilizenucleic acids. Such reagents are the basis for analytical procedures and diagnostic kits 15 that are commercially available.

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

When reagent molécules are subjected to stress due to fluctuating températures20 and/or high température conditions, they may rapidly lose their biological activity. Thus, commercial products that contain these molécules 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 25 2 012935 molécules may be damaged.

To obviate the problem of stability, some technical solutions hâve been proposedwhich consist of coupling the bioactive molécules to a solid phase (Kochanowska IE,Rapak A, Szewczuk A, "Effect of pretreatment of wells in polystyrène plates onadsorption of some human sérum proteins," Arch Immunol Ther Exp (Warsz) 199442:135-9; Ishikawa E, Hamaguchi Y, Imagawa M, Inada M, Imura H, Nakazawa N,Ogawa H, "An improved préparation of antibody-coated polystyrène beads for sandwichenzyme 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 antigenconcentration and stability in ELISA," J Immunol Methods, 1986 Nov 20 94:1-2 137-44;Kakabakos SE, Livanlou E, Evangelatos GP, Ithakissios DS,"Immobilization ofimmunoglobulins onto surface-treated and untreated polystyrène beads forradioimmunoassays," Clin Chem, 1990 Mar 36:3 492-6; Ansari AA, Hattikudur NS,Joshi SR, Medeira MA, "ELISA solid phase: stability and binding characteristics,"JImmunol 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 dénaturation ofribonucléase Tl. A DSC Study," J.Thermal Analysis, 38 (1992) 2791-2802.)

Conjugation of the molécule to a solid substrate permits the storage of product in a non-hydrated form. This approach dépends upon a Chemical interaction between the reagentand the solid phase and does not allow the subséquent solubilization of the reagent.However, proteins coupled to solid phases hâve a good stability only when stored at lowtempératures (2°C to 8°C) and may be rapidly inactivated at higher températures. 3 012935

Another System typically utilized for molécule storage is lyophilization (Vallet G.,"Lyophilization or freeze drying. An idéal method of préservation but also one ofscientifïc search for the phytotherapeutic constituées of "Agaricus campestris" or Parismushroom," Presse Med, 1966 Mar 26 74:16 846; List PH, "Lyophilization, freezedrying," 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 hemoglobinin érythrocytes after their prolonged préservation in a frozen State and lyophilization,"Probl Gematol Pereliv Krovi, 1970 Nov 15:11 28-32). This method, based on watersublimation starting from ice, cannot be applied to molécules which hâve a complexquatemary structure, since the method induces conformational changes that may resuit inloss of biological activity. An additional problem is that the optimal storage températurefor lyophilized material is generally very low, for example, -20°C for thermolabileenzymes, which can be difficult to achieve and maintain in certain clinical situations.

Therefore, there is a need to develop a method for preparing stabilizeddehydrated, preferably anhydrous molécules, for reagent or therapeutic purposes, that arerésistant to dégradation due to unfavorable environmental conditions. In particular,proteins having the capability to retain biological activity upon exposure to theseconditions would be useful for various applications in analytical and diagnostic analysis. 4 012935

SUMMARY OF THE INVENTION

The présent invention provides for a method of preparing stabilized dehydrated,preferably anhydrous, bioactive molécules whereby déhydration is accomplished byévaporation rather than sublimation, thereby avoiding the harsh, potentially deactivatingconditions associated with lyophilization. In particular, the bioactive molécule to bestabilized, in an aqueous environment, is combined with a detergent, such as a non-ionicdetergent, which, without being bound to any particular theory, forms a micelle thatpréserves the configuration of the bioactive molécule during the évaporation process.

BRIEF DESCRIPTION QF THE FIGURES

Figure 1A-B. (A.) Détermination of circulating CD4 positive lymphocytes byflow cytometry using FITC-labelku anti-CD4 monoclonal antibody in liquid form. Thepercentage of CD4 positive cells was found to be 32.8 percent. The resolution index wascalculated to be 7.33. (B) Détermination of circulating CD4 positive lymphocytes byilow cytometry using FITC-labelled anti-CD4 monoclonal antibody in anhydrous formaccording to the invention. The percentage of CD4 positive cells was found to be 33.1percent. The resolution index was found to be 7.30.

Xpos — xneg Résolution index ! ÿ z x yl(SDpos)2 + (SD neg)2

Figure 2. Variation of the resolution index over time as a function of storage température of anhydrous, FITC-labelled anti-CD4 monoclonal antibody according to the invention. 5 012935

DETAILED DESCRIPTION OF THE INVENTION

The présent invention describes a method for preparing a biological moléculecapable of retaining biological activity when stored under unfavorable conditions.

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

Various molécules may be used as the bioactive molécules to be stabilizedaccording to the présent invention. They include, but are not limited to, monoclonal orpolyclonal antibodies, proteins, enzymes, polypeptides, nucleic acids, polysaccharidesand lipids. These bioactive molécules may optionally be conjugated to other molécules,for example fluorochromes, enzymes, colloids, isotopes, chemoluminescent substances,or other molecular ligands or labels known in the art.

Preferably, the detergent has minimal de-acti/ating or denaturing effect on thebioactive molécule to be stabilized. In preferred, non-limiting embodiments of theprésent invention, the detergent is a non-ionic detergent. Détergents which may be usedin the présent 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-P-D-glucopyranoside;Decyl-p-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; 6 012935 1 heptyl-P-D-glucopyranoside; heptyl-p-D-thioglucopyranoside; hexyl-β-ϋ-glucopyranoside; lauryldimethylamine oxide; lauryl sulfate; octanoyl-N-methylglucamide; nonaoyl-N-methylglucamide; decanoyl-N-methylglucamide; nonyl-β-D-glucopyranoside; NP-40; Octyl-P-D-glucopyranoside; octyl-p-D-maltopyranoside; 5 octyl-β-ϋ thiogalactopyranoside; octyl^-D-thioglucopyranoside; taurocholic acid; taurodehydrocholic acid; taurodeoxycholic acid; Triton X-100; Triton X-114; TWEEN20; TWEEN 80; N-octyl-N,N-dimethyl-3-ammonio-l-propanesulfonate; N-decyl-N,N-dimethyl-3 -ainmonio-1 -propanesulfonate; N-dodecyl-N,N-dimethyl-3 -ammonio-1 -propanesulfonate; and N-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate N- 10 hexadecyl-N,N-dimethyl-3 -ammonio-1 -propanesulfonate.

The amount of detergent added to the molécule to be stabilized is dépendent upon the type of detergent used and molécule to be stabilized. Knowledge of such amounts iswithin the skill of one in the art. For example, but not by way of limitation, theconcentraion of bioactive molécule may be between 0.1 picogram and 100 milligram, 15 preferably between 1 picogram and 1 milligram, per milliliter of solution. Preferably, thebioactive molécule to be stabilized is in an aqueous environment (for example, but not byway of limitation, phosphate buffered saline) and the detergent is présent at aconcentration above its critical micelle concentration, where the critical micelle

concentrations for détergents may be found in standard technical reference materials. A 20 non-limiting list of critical micelle concentrations may be found in P. Mukerjee, K. J.

Mysels, Critical Micelle Concentrations of Aqueous Surfactant Systems, NSRDS-NBS, 36 (1971). This concentration is preferably achieved prior to the évaporation process. In spécifie non-limiting embodiments of the invention, the detergent is présent in an amount 25 7 012935 which solubilizes the compound to be stabilized. According to preferred non-limitingembodiments of the invention, the concentration of detergent is less than or equal toabout 1.0 % weight/volume ("w/v").

In an embodhnent of the présent invention, Tween 20 is mixed with the bioactivemolécule to a concentration preferably, but not by way of limitation, greater than about0.055 mM, or greater than about 0.06 mM, or greater than about 0.07 mM, or greater thanabout 0.08 mM. Preferably, Tween 20 is added to a final concentration of between0.008% 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 détergents may be used. Preferably, at least 50 mM ofNa+ or an équivalent ion is also présent.

After the molécule to be stabilized is mixed with the detergent, it is dried such thatwater in the mixture evaporates. Evaporation may be performed at températures whichare preferably lower tnan the température at which the molécule to be stabilizeddénaturés and/or is inactivated. Preferably évaporation is performed at ambienttempératures (10-45°C, preferably 15-25°C) although lower or higher températures (forexample, 4-50°C) may be used provided that the compound to be stabilized is notsignificantly inactivated. Evaporation is preferably performed under low atmosphericpressure 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 theamount of time required for évaporation. The size and type of the final container duringévaporation may also effect the évaporation.

The évaporation step may be performed by placing the dispensed mixtures into aconcentrator instrument. The concentrator instrument may, for example but not by way 8 012935 of limitation, be a centrifuge connected to a vacuum pump and having a cool trap for steam condensation.

The évaporation step can be performed not only using the described apparatus, butalso without applying centrifugation.

The amount of time required for évaporation dépends on the volume ofbioactivemolecule/detergent solution, the size of the évaporation vessel, the température andpressure, factors which may be controlled using techniques known in the art. Thecompletion of the process, when adéquate déhydration has been achieved, can also bedetermined using standard laboratory techniques.

The évaporation 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 désirable toperforai the various steps in the absence of-light.

Preferably évaporation is continued to dehydrate the compound, more preferablyuntil the compound is essentially anhydrous.

Prior to use, it may optionally be désirable to remove the detergent from thecomposition resulting after évaporation. Such removal may preferably be performedafter storage and relatively immediately prior to use. Removal may be performed usingstandard techniques, such as dialysis.

Without being bound by any particular theory, it is believed that evaporating themixture generates a water shield around the molécule that imparts a higher stability to themolécule. If the molécule is a protein, dehydrated, and preferably anhydrous proteins areobtained by the water évaporation step. The présent invention is superior tolyophilization, since it avoids the température stress created during the water sublimation, 9 012935 1 which could cause the protein to dégradé.

The methods of the présent invention allows the storage of the stabilized bioactive molécule at elevated températures which must be lower than the dénaturation températureof the protein. In addition, the procedure allows an increase in the product shelf life, 5 which may exceed more than 8 years when the product is correctly stored.

Stabilized bioactive molécules according to the invention may be used for diagnostic or therapeutic purposes, as appropriate. The présent invention provides fordiagnostic or therapeutic compositions comprising such stabilized bioactive molécules, aswell as for diagnostic kits comprising such stabilized molécules. 10 EXAMPLE 1. The stabilization of murine monoclonal antibody conjugated withfluorescein isothiocyanate (FITC). A monoclonal antibody specuiu for the CD4 molécule and conjugated to afluorochrome (fluorescein- FITC) is used as the starting material to generate the molécule 15 of the invention (clone EDU-2 IgG2a isotype). The CD4 molécule is expressed on a monocytes and a subset of T lymphocytes. The CD4 monoclonal is commonly used todétermine the number and the percentage of CD4+ T cells in the blood by flow cytometry.

For the présent example, the method of the présent invention fulfilled dual goals 20 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 sérum albumin (BSA) 1% weight on 25 10 012935 volume (w/v), natrium azide NaN3 0.2% w/v, pH 7.2-7.4. To this solution, the detergentis added (Tween 20) to a final concentration of 0.1% w/v. After mixing at roomtempérature away from direct light exposure, 10 pL of solution is dispensed intopolystyrène test tubes having 12 mm internai diameter. The tubes are then placed insidea concentrator instrument which consists in a centrifuge connected with a vacuum pumpand a cool trap for steam condensation. The tubes are subjected to a 4 hour déhydrationprocess without direct light exposure due to avoid dégradation of the photosensitivefluorochrome. 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 evaluatedfor the maintenance of biological activity. The resolution index of the anhydrousmonoclonal antibody, which is an indicator of the efficiency of the binding betweenfluorochrome and protein, was also examined. The results of the comparison are reportedin Figure 1 and Table I.

The anhydrous monoclonal antibody was also tested for its ability to preservebinding and fluorescence using an accelerated stability test at various high températures.The anhydrous monoclonal antibody were stored at the following three températures: 4°C, 37°C and 45°C. At various time points, aliquots were removed from the threetempérature conditions from the incubators and tested with a control reference sample byflow cytometry. From such analysis decay curves based upon the resolution index hâvebeen drawn (see Figure 2 of Table II). 11 012935

Key words: protein storage, protein stabilization, high température évaporation,surfactants, détergents, anhydrous, dry form.

Claims (11)

12 012935 CLAIMS

1. A method for preparing a dehydrated stable bioactive molécule capable ofretaining biological activity when stored under unfavorable conditions comprisingmixing a bioactive molécule with a detergent to create a mixture and evaporatingthe mixture to obtain the dehydrated stable molécule.

2. The method according to claim 1 wherein the bioactive molécule is a protein.

3. The method according to claim 2 wherein the protein is a monoclonal antibody.

4. The method according to claim 3 wherein the monoclonal antibody is conjugatedto a détection molécule.

5. The method according to claim 4 wherein the détection molécule is a fluorochrome.

6. The method according to claim 5 wherein the fluorochrome is fluoresceinisothiocyanate (FITC).

7. The method according to daims 1, 2, 3, 4, or 5, wherein the detergent is a non-ionic detergent.

8. The method according to claim 1 further comprising dispensing the mixture intotest tubes prior to évaporation.

9. The method according to claim 1 wherein the mixture is evaporated by the using aconcentrator instrument.

10. A stabilized molécule prepared according to any of daims 1-9.

11. A diagnostic kit comprising a stabilized molécule according to claim 10.