WO2004028259A2 - Procedde de stabilisation de molecules bioactives - Google Patents

Procedde de stabilisation de molecules bioactives Download PDF

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Publication number
WO2004028259A2
WO2004028259A2 PCT/EP2003/010699 EP0310699W WO2004028259A2 WO 2004028259 A2 WO2004028259 A2 WO 2004028259A2 EP 0310699 W EP0310699 W EP 0310699W WO 2004028259 A2 WO2004028259 A2 WO 2004028259A2
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WO
WIPO (PCT)
Prior art keywords
molecule
stabilized
detergent
molecules
mixture
Prior art date
Application number
PCT/EP2003/010699
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English (en)
Other versions
WO2004028259A3 (fr
Inventor
Silva Barbesti
Francesco Cipriani
Original Assignee
Bio-D S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bio-D S.R.L. filed Critical Bio-D S.R.L.
Priority to AU2003271642A priority Critical patent/AU2003271642A1/en
Priority to AP2005003253A priority patent/AP2005003253A0/xx
Publication of WO2004028259A2 publication Critical patent/WO2004028259A2/fr
Publication of WO2004028259A3 publication Critical patent/WO2004028259A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4

Definitions

  • 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.
  • 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).
  • reagent molecules are subjected to stress due to fluctuating temperatures and/or high temperature conditions, they may rapidly lose their biological activity.
  • 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.
  • 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 (Nallet G., "Lyophilization or freeze drying.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • bioactive 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.
  • the detergent has minimal de-activating or denaturing effect on the bioactive molecule to be stabilized.
  • 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)-dime1hyl-ammonio-2-hydroxi- 1 - propanesulfonate; chenodeoxycholic acid; cholic acid; Decyl- ⁇ -D-glucopyranoside;
  • Decyl- ⁇ -maltoside Deoxicholic acid; Digitonin; dodecyl- ⁇ -D-maltoside; N-dodecyl-N- N-dimethylglycine; bis-(2-Ethylexyl)sodiumsulfosuccinate: polyoxyethylene (10) dodecyl ether, polyethylene glycol lauryl ether; glycocholic acid; glycodeoxycholic acid; heptyl- ⁇ -D-glucopyranoside; heptyl- ⁇ -D-thioglucopyranoside; hexyl- ⁇ -D- glucopyranoside; lauryldimethylamine oxide; lauryl sulfate; octanoyl-N- methylglucamide; nonaoyl-N-methylglucamide; decanoyl-N-methylglucamide; nonyl- ⁇ -
  • octyl- ⁇ -D thiogalactopyranoside octyl- ⁇ -D-thioglucopyranoside; taurocholic acid; taurodehydrocholic acid; taurodeoxycholic acid; Triton X-100; Triton X-l 14; TWEEN 20; TWEEN 80; N-octyl-N,N-dimemyl-3-ammonio-l -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-ammomo-l-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.
  • the concentraion of bioactive molecule maybe between 0.1 picogram and 100 milligram, preferably between 1 picogram and 1 milligram, per milliliter of solution.
  • 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 maybe found in P.
  • 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").
  • 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.
  • 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.
  • at least 50 mM of Na+ or an equivalent ion is also present.
  • 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.
  • 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.
  • evaporation is continued to dehydrate the compound, more preferably until the compound is essentially anhydrous.
  • removal may preferably be performed after storage and relatively immediately prior to use. Removal may be performed using standard techniques, such as dialysis.
  • 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.
  • 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.
  • 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.
  • PBS saline phosphate buffer
  • BSA bovine serum albumin
  • natrium azide NaN3 0.2% w/v pH 7.2-7.4.
  • the detergent is added (Tween 20) to a final concentration of 0.1% w/v.
  • 10 ⁇ L 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 fluo
  • 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). Keywords: protein storage, protein stabilization, high temperature evaporation, surfactants, detergents, anhydrous, dry form.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un procédé de préparation de molécules stabilisées, sous forme déshydratée. Le procédé permet la préparation de molécules, notamment de protéines et plus particulièrement d'anticorps, que l'on peut stocker dans des conditions environnementales défavorables, à savoir lors de fluctuations de la température et à des températures élevées.
PCT/EP2003/010699 2002-09-24 2003-09-23 Procedde de stabilisation de molecules bioactives WO2004028259A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003271642A AU2003271642A1 (en) 2002-09-24 2003-09-23 A method for stabilizing bioactive molecules
AP2005003253A AP2005003253A0 (en) 2002-09-24 2003-09-23 A method for stabilizing bioactive molecules.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000032A ITBA20020032A1 (it) 2002-09-24 2002-09-24 Metodo per stabilizzare le proteine per la conservazione ad alte temperature.
ITBA2002A000032 2002-09-24

Publications (2)

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WO2004028259A2 true WO2004028259A2 (fr) 2004-04-08
WO2004028259A3 WO2004028259A3 (fr) 2004-05-27

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AP (1) AP2005003253A0 (fr)
AU (1) AU2003271642A1 (fr)
IT (1) ITBA20020032A1 (fr)
OA (1) OA12935A (fr)
WO (1) WO2004028259A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010142A1 (fr) * 1988-04-29 1989-11-02 Igen, Inc. Systeme d'interaction comprenant une phase aqueuse dispersee stabilisee par tensio-actif contenant un anticorps ou un fragment d'anticorps
WO1995025119A1 (fr) * 1994-03-16 1995-09-21 Mallinckrodt Medical, Inc. Stabilisation de peptides et de proteines dans le but d'une utilisation radiopharmaceutique
EP0988861A1 (fr) * 1998-08-17 2000-03-29 Pfizer Products Inc. Composition de protéines stabilisée
EP1136068A2 (fr) * 2000-03-21 2001-09-26 Jcr Pharmaceuticals Co., Ltd. Poudre de peptide à activité physiologique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010142A1 (fr) * 1988-04-29 1989-11-02 Igen, Inc. Systeme d'interaction comprenant une phase aqueuse dispersee stabilisee par tensio-actif contenant un anticorps ou un fragment d'anticorps
WO1995025119A1 (fr) * 1994-03-16 1995-09-21 Mallinckrodt Medical, Inc. Stabilisation de peptides et de proteines dans le but d'une utilisation radiopharmaceutique
EP0988861A1 (fr) * 1998-08-17 2000-03-29 Pfizer Products Inc. Composition de protéines stabilisée
EP1136068A2 (fr) * 2000-03-21 2001-09-26 Jcr Pharmaceuticals Co., Ltd. Poudre de peptide à activité physiologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG M Z ET AL: "A NEW STRATEGY FOR ENHANCING THE STABILITY OF LYOPHILIZED PROTEIN: THE EFFECT OF THE RECONSTITUTION MEDIUM ON KERATINOCYTE GROWTH FACTOR" PHARMACEUTICAL RESEARCH, NEW YORK, NY, US, vol. 12, no. 10, 1995, pages 1447-1452, XP009007833 ISSN: 0724-8741 *

Also Published As

Publication number Publication date
WO2004028259A3 (fr) 2004-05-27
ITBA20020032A1 (it) 2004-03-25
AU2003271642A8 (en) 2004-04-19
AP2005003253A0 (en) 2005-03-31
OA12935A (en) 2006-10-13
AU2003271642A1 (en) 2004-04-19

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