METHOD AND PREPARATIONS FOR STABILIZING BIOLOGICAL MATERIALS BY DRYING ...

METHOD AND PREPARATIONS FOR STABILIZING BIOLOGICAL MATERIALS BY DRYING METHODS WITHOUT FREEZING

TECHNICAL FIELD

[0001] The present invention concerns preparations and processes for stabilizing biological materials by means of drying processes without freezing. Specially selected mixtures of sugars and amino acids and derivatives thereof as well as of various amino acids and derivatives thereof are described which can be used to achieve a particularly advantageous stabilization of peptides, proteins, glycoproteins, antibodies and similar substances after producing dry partially amorphous products by drying processes in which freezing is not employed.

STATE OF THE ART

[0002] The production of storage-stable (in particular at room temperature) preparations of biologically active and therapeutic substances such as peptides, proteins, glycoproteins, nucleotides, plasmids, cell fragments, viruses etc. for diagnostic and therapeutic purposes is nowadays of great and continually increasing importance.

[0003] Various processes and formulations for producing dry biologically or therapeutically active material have been described. Dry material is understood as substances and mixtures of substances which have a maximum residual moisture of 8% (g/g), preferably of at most 4% (g/g) particularly preferably of at most 2%. Freeze drying processes are widespread but have disadvantages [F. Franks, Cryo Lett. 11, 93-110, (1990); M. J. Pikal, Biopharm. 3 (9), 26-30 (1990); M. Hora, Pharm. Research 8 (3), 285-291 (1992); F. Franks, Jap. J. Freezing Drying 38, 15-16, (1992)]. They consume large amounts of energy, require the use of refrigerants (Frigens) some of which are harmful, and take a long time. For numerous substances, in particular proteins, the step of freezing which is necessary for freeze drying is damaging i.e. destabilizing. This process cannot therefore be used at all for some biological materials.

[0004] Alternatives to freeze drying for producing dry protein preparations are processes which dry the material by the application of heat and or a vacuum [F. Franks, R. M. H. Hatley; Stability and Stabilization of Enzymes; Eds. W. J. J. van den Teel, A. Harder, R. M. Butlaar, Elsevier Sci. Publ. 1993, pp. 45-54; B. Roser, Biopharm. 4 (9), 47-53 (1991); J. F. Carpenter, J. H. Crowe, Cryobiol. 25, 459-470 (1988)]. Examples of this are vacuum drying with or without the application of an increased temperature, spray-drying processes in various modifications including the combined application of a vacuum and spraying procedure as well as drum drying and other thin layer drying processes.

[0005] Preparations are described in J. F. Carpenter, J. H. Crowe, Biochemistry 28, 3916-3922 (1989); K. Tanaka, T. Taladu, K. Miyajima, Chem. Pharm. Bull. 39 (5), 1091-94 (1991), DE-C-3520228, EP-B-0229810, WO 91/18091, EPB-0383569, US 5,290,765 which contain sugar or sugar-like substances. In the production of dry sugar preparations the following disadvantages and problems have been found in the processes described in the state of the art: The production of really adequately dry sugar preparations is not possible without the use of a significant amount of energy. This applies particularly to preparations in the final container. It

is possible to apply warmth/heat for this but this must be judged to be extremely critical with regard to the stability of the biological materials used. Alternatively in order to achieve adequate drying with a low heat input, drastically increased process times or extremely thin layer thicknesses can be used. Both procedures do not lead to the goal. Long process times are economically extremely unfavourable, moreover the long residence time of an active biological substance in a matrix that is only slowly depleted of water is destabilizing and thus also critical. The drying of thin layer thicknesses does not lead in many cases to an economically viable yield of product i.e. only minimal amounts of product are obtained per unit of time and/or drying area. In addition the processing of biological materials on very large open drying areas can hardly be accomplished with the sterility that is often necessary for the pharmaceutical and diagnostic application.

[0006] Drying processes which proceed by means of a vacuum at a temperature that is lower than or slightly above room temperature are milder. However, in many cases it is practically hardly possible to produce dry storage-stable sugar preparations. When sugar solutions are dried increasingly viscous, thick pastes are formed. The residual amount of water or residual moisture remaining in these materials cannot be removed within an economically reasonable period, in many cases the drying comes to a standstill at a high level which is not suitable for stabilization. The degradation manifests itself for example in a decrease in the activity of the stored material, in the formation of aggregation products or by the occurrence of degradation products of a lower molecular weight. A suitable low residual water content for the stabilization of proteins etc. can be identified on the basis of physical parameters. It follows from the literature cited above that preparations suitable for stabilizing proteins etc. should have a glass-like i.e. an amorphous structure the glass transition temperature of which lies above the envisaged storage temperature. The glass transition temperature is that temperature at which an amorphous solid body changes from the glass state into the thick viscous state and vice versa. Drastic changes in viscosity occur in this process and concomitantly in the diffusion coefficients and the kinetic mobility of the proteins and other molecules. Physical parameters such as hardness and modulus change as well as the thermodynamic functions of state: volume, enthalpy and entropy. The glass transition temperature of for example a material containing sugar and its residual water content are linked physically to one another in such a way that increasing amounts of residual water lead to reduced glass transition temperatures and vice versa. Thus the measurement of the glass transition temperature e.g. by differential scanning calorimetry (DSC) can be used to deduce whether a preparation has a suitable residual water content for stabilization and, as described above, whether a drying process is successful or not. In addition to the determination of the glass transition temperature by means of DSC, the presence of amorphous structures can also be proven by means of X-ray diffraction investigations and optical and electron microscopic observations.

[0007] Therefore it is desirable to provide a stabilizing matrix for biologically or pharmaceutically active materials with a glass transition temperature that lies above the storage temperature, which contains a low residual moisture and processes for the cost-effective production of such stabilizing matrices.

[0008] Description of the Invention:

[0009] Surprisingly it was found that the addition of zwitterions with apolar residues to materials containing carbohydrates can change their drying properties in such a positive manner that materials which previously dried poorly and accordingly did not have adequate stabilizing properties could now be dried very rapidly and produced an excellent stability of the biologically and in particular therapeutically active materials formulated therein.

[0010] Furthermore it was surprisingly found that carbohydrate-free formulations composed of mixtures of particular zwitterions could also be dried very rapidly and had very good stabilizing properties. In this case a zwitterion with a polar residue must be used together with a zwitterion with an apolar residue. Such zwitterions are preferably aminocarboxylic acids and derivatives thereof and particularly preferably pharmaceutically acceptable amino acids. Zwitterions are understood as low-molecular compounds whose molecular weight is below 10 kDa and preferably below 5 kDa. Processes are described which, without the application of a high temperature i.e. at room temperature, allow preparations according to the invention to be dried in such a way that suitable glass transition temperatures are reached for preparations for stabilizing biologically and in particular therapeutically active substances. Biologically active substances are, in addition to therapeutically active substances, also those which are used in biotechnological processes such as e.g. fermentation. As well as those substances which are used for example in plant protection or as an insecticide. Such biologically and in particular therapeutically active materials can for example be selected from one or several substances of the groups proteins, peptides, glycoproteins, lipoproteins, enzymes, coenzymes, biological membranes, antibodies, antibody fragments, viruses, viral components, vaccines, DNA, RNA, PNA, plasmids, vectors, pheromones, biological therapeutics and diagnostics and derivatives thereof. Biologically active substances are not understood to include foods as such.

[0011] The particular advantages of the preparations and processes described here are:

[0012] that a freezing is avoided during the drying

[0013] the drying can be carried out with freeze-drying plants that are already available in the chemical-pharmaceutical industry without any retrofitting

[0014] filling into commercial containers e.g. glass bottles which is particularly advantageous for an aseptic production can be retained without change

[0015] process times are of the same order of magnitude as freeze-drying processes and much less

[0016] toxicologically acceptable auxiliary substances can be used

[0017] all quantities of energy necessary for freezing can be saved and the use of environmentally harmful refrigerants can be drastically reduced

[0018] the products obtained are readily visible "cakes" that can be rapidly dissolved again

[0019] since a partially amorphous state is rapidly attained, the biological material is degraded less than by the processes described in the state of the art.

[0020] It should be noted that the particular advantages of the formulations described here of particular mixtures of sugars and amino acids as well as of particular mixtures of at least 2 amino acids are also effective when they are used within the framework of other drying processes which avoid freezing. The accelerated drying effect of the additives as well as the property of the preparations to form amorphous or partially amorphous systems equally applies to spraydrying, drum-drying etc.

[0021] An essential feature is that significant amounts of amorphous materials are present as detected by DSC and/or X-ray structural analysis or other suitable methods and that the preparations do not have a completely crystalline character. Crystalline preparations are not suitable for achieving an adequate stability for sensitive biological substances. Completely amorphous preparations are suitable for stabilization and are thus in principle according to the invention but partially amorphous preparations are especially so.

DESCRIPTION OF THE FIGURES

[0022] FIG. la: glass transition temperatures of individual maltose-L-phenylalanine mixtures

[0023] FIG. lb: residual water content of the individual maltose-L-phenylalanine mixtures

[0024] FIG. 2: powder diffractograms of vacuum-dried

[0025] (a) phenylalanine (water content 1.2%/crystalline),

[0026] (b) maltose (water content 4.0%, Tg=50° C.) and

[0027] (c) phenylalanine and maltose prepared in a manner according to the invention (water content 0.7%, Tg=88° C).

[0028] The diffractograms were recorded with a conventional instrument (Phillips 1730 X-ray) and associated software. The measuring temperature is 25° C, the angular resolution (29) 0.05°. Measuring conditions: 1 s per angle at 40 kV tube voltage and 40 mA current strength.

[0029] FIG. 3: Time course of the

[0030] (a) residual water content and the

[0031] (b) glass transition temperature of a maltose/
phenylalanine preparation according to the inven-
tion.

DETAILED DESCRIPTION OF THE
INVENTION

[0032] The invention is exemplified by 13 examples and 10 comparative examples and is elucidated in the following. In this process formulations and processes were found which drastically improve and accelerate the drying of materials containing sugar by means of vacuum-drying and are suitable for stabilizing relevant therapeutic and diagnostic biological materials. Furthermore completely novel compositions are shown which fulfil the purpose of stabilization while retaining the optimized drying characteristics.

[0033] These compositions preferably contain either at least one zwitterion with an apolar residue (e.g. an amino acid such as phenylalanine) and sugar in which the glass transition temperature of the sugar is considerably increased