FR2881139A1 - COMPOSITION FOR THE LYOPHILIZATION OF PROTEINS - Google Patents

Abstract

The present invention relates to the use of liquid compositions in the context of the implementation of the lyophilization process on proteins, for the purpose of stabilizing these proteins, said compositions comprising: a filler having a temperature of collapse between -18 degree C and 0 degree C, - a stabilizing agent, - a buffer solution, - and, if appropriate, a nonionic surfactant.

Description

COMPOSITIONS FOR THE LYOPHILIZATION OF PROTEINS

  The present invention relates to the use of liquid compositions in the context of the implementation of the lyophilization process on specific proteins, previously diluted 5. in these compositions, for the stabilization of these proteins in the dry state after freeze-drying and, if appropriate, in the liquid state after rehydration of the lyophilizate obtained.

  Protein stability is generally insufficient to allow their distribution and use in liquid form [1]. Thus, to ensure their stability over storage periods of several months, the proteins are lyophilized. The vacuum lyophilization process involves three essential steps: (1) freezing which converts most of the water present to ice; (2) primary drying which allows the sublimation of the ice after decreasing the pressure and increasing the temperature; (3) secondary desiccation which allows the desorption of the remaining unfrozen water by increasing the temperature. Freeze-dried products, characterized by a very low residual water content (less than 0.05 g of water per g of product) have several advantages: a lifetime of several years, storage at room temperature or at +4 C thus facilitating their distribution and handling as well as instant rehydration properties.

  However, even if the lyophilization process improves the stability of the protein, it generates different stresses that can denature the protein. The addition of excipients, such as sugars, polyols or amino acids, is therefore necessary to protect the protein during the freezing and dehydration steps. Other excipients, such as polymers, salts, ions, can also be used for their filler or buffering properties. A wide variety of molecules, alone or in combinations, at various concentrations, providing effective protection has been identified in the literature [2]. Thus, the formulation step is complex and the development of new freeze-dried proteins remains a long and empirical step.

  Recently, Carpenter et al. [3] stated a formulation rule based on the combination of four compounds: (1) a buffering agent not crystallizing during the freezing step; and / or (2) a stabilizer preserving the conformation of the protein in an amorphous solid matrix (most often a disaccharide such as sucrose or trehalose), and / or (3) a stabilizing agent for stability physical product (mannitol, glycine, starch hydroxylethyl or bovine serum albumin), and / or 4) a nonionic surfactant to reduce protein aggregation. Nevertheless, from this rule, multiple combinations remain possible, because of the diversity of the molecules and their variable concentration.

  The formulation step and the lyophilization process are closely related to each other. The development of the process depends on the properties of the chosen formulation and conversely any variation of the process may change the physical state of the formulation. In addition, freeze-drying is a time-consuming and expensive process because of the high dehydration times (cycles of one to several days). Today, it therefore becomes essential to take into account the criterion of improving the productivity of the process from the formulation stage by selecting appropriate excipients.

  Considering the conduct of the freeze-drying process, the primary drying, the longest step of the process, must be carried out at the highest possible product temperature (Tmax) resulting from the operating conditions applied (shelf temperature and chamber pressure).

  If the temperature of the product is higher than Tmax, the material will lose the porous structure formed during the freezing step and collapse on itself, this phenomenon is called collapse or collapse [4]. Generally collapsed products have higher residual water content, longer rehydration times. Although the biological activity of the protein can be preserved, their non-compliant visual appearance prevents their commercialization. The value of Tmax, associated with the collapse temperature (Tcoll) depends on the physical properties of the frozen product. If the material is essentially crystalline after freezing, Tmax will correspond to the eutectic temperature (Te) of the solute. On the other hand, if the material is in the amorphous solid state after freezing, Tmax will be associated with the glass transition temperature of the maximum cryo-concentrated phase (Tg '). In the case of complex materials, including tissues or cells for example, the value of the collapse temperature Tcoll (and therefore Tmax) will be between Te or the melting temperature and Tg '[5]. Differential enthalpic analysis and cryo-microscopy are the two methods generally used to characterize the physical behavior of pharmaceutical formulations [4, 6-9]. Cryo-microscopy makes it possible to visually determine the collapse temperature (Tcoll), ie the temperature from which the sublimation of the ice is accompanied by the collapse of the dry part [10].

  The main purpose of the present invention is to provide liquid compositions for lyophilization: - not having any excipient of animal origin, - ensuring a good productivity of the freeze-drying process (short cycle and robustness of the formula), preserving the activity of proteins with pharmaceutical applications during storage in the freeze-dried state (extreme conditions: 6 months at 25 ° C.), and, if appropriate, during storage in the liquid state after rehydration (conditions extremes: 3 months at 4 C).

  Seeking to develop a rational approach for the development of freeze-dried formulations in connection with the productivity of the process, the inventors have chosen to consider the glass transition temperature (Tg ') and the collapse temperature (Tcoll) as selection criteria. .

  The inventors have thus demonstrated that the compositions comprising: a filler having a collapse temperature of between 18 ° C. and 0 ° C., a stabilizing agent, a buffer solution, and, if appropriate, a surfactant nonionic agent, in proportions such that the collapse temperature of the composition thus obtained is between -16 C and -10 C, allowed to achieve the fixed objective.

  As such, the present invention relates to the use of a liquid composition in the context of the implementation of the lyophilization process on specific proteins, previously diluted in said composition, for the stabilization of these proteins to the dry state after lyophilization, and, if appropriate, in the liquid state after rehydration of the lyophilizate obtained, said composition having a crystalline or amorphous solid structure in the dry state after lyophilization, and comprising: - about 40 g / l to 60 g / l g / L of a filler having a collapse temperature of -18 ° C to 0 ° C, - about 10 g / L to 20 g / L of a stabilizing agent, -about 10 mM to 20 mM of a buffer solution, said buffer solution not crystallizing during the freezing of said composition, and, if appropriate, about 0.1 g / l to 0.4 g / l of a nonionic surfactant, said composition liquid being such that: * the mass ratio of ag When the said composition has a predominantly crystalline structure after freeze drying, the load ratio on the stabilizing agent is greater than 3: 1, the ruble ratio of the stabilizing agent on the protein is greater than 1: 1. contains no excipient of animal origin, * it has a collapse temperature between -16 C and -10 C.

  By the term "filler", in the foregoing and the following, is meant any agent imparting mechanical strength to the final product, and contributing to the improvement of the appearance of the freeze-dried product and the robustness of the material by reducing the risk of collapse of the product during the process.

  By the term collapse temperature, in what precedes and what follows, is meant the temperature from which the sublimation of the ice is accompanied by a loss of structure of the dry region of the product. Advantageously, this temperature is measured by cryomicroscopy according to the methods described in the literature [5, 10].

  By the term stabilizing agent, in the foregoing and the following is meant any compound protecting the protein during lyophilization and storage. It must be at least in the amorphous solid state (vitreous state) after freezing and dehydration, and be able to replace the water by forming hydrogen bonds with the protein.

  Advantageously, as indicated above, the abovementioned buffer solution is such that it does not crystallize during the freezing of said composition (especially at product temperatures below -35 ° C., for example between -45 ° C. and -35 ° C. VS).

  By the expression "non-ionic surfactant", in what precedes and what follows, is meant any compound reducing the adsorption of the protein on a surface (walls of the flasks, stopper, air-water interface, water-ice interface, by example); especially when the protein is present in very low concentration.

  The subject of the invention is more particularly the aforementioned use of a composition as defined above, characterized in that the filler is chosen from: polyols, such as mannitol or inositol, preferably mannitol, or amino acids, such as glycine or alanine, preferably glycine, when said compositions have a predominantly crystalline structure after lyophilization, or polysaccharides, especially those having an equivalent dextrose (DE) less than 10, such as maltodextrin, dextran, or hydroxyethyl starch (also referred to as HES for hydroxyethyl starch), preferably maltodextrin, when said compositions exhibit an amorphous solid structure after lyophilization.

  The above-mentioned equivalent dextrose (DE) is a polysaccharide behavior index that varies as the inverse of the molar mass. A DE maltodextrin of between 6 and 10 has a molar mass of about 100,000 g / mol.

  The invention relates more particularly to the aforementioned use of a composition as defined above, characterized in that the stabilizing agent is chosen from: polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA), polymers having a molecular weight of less than 30 kDa, preferably PVP, when said compositions have a predominantly crystalline structure after lyophilization, or sucrose, trehalose, maltose or lactose, preferably sucrose, when said compositions exhibit an amorphous solid structure after lyophilization.

  The invention more particularly relates to the aforementioned use of a composition as defined above, characterized in that the surfactant is selected from Tween 80 (Polysorbate 80: Polyoxyethylene sorbitan monooleate), Tween 20 ( Polysorbate 20: Polyoxyethylene sorbitan monolaurate), Triton X-100 (Octyl phenoxy polyethoxyethanol), or Brij 35 (Polyethylene glycol dodecyl ether), preferably Tween 80.

  The invention relates more particularly to the aforementioned use of a composition as defined above, characterized in that the buffer solution is a solution of Tris HCl (pH 7.8), or potassium phosphate, preferably a solution of Tris-HC1.

  Advantageously, the liquid compositions as defined above, having a predominantly crystalline structure in the dry state after lyophilization, comprise: about 40 g / l to 60 g / l of a filler as defined above, about 10 g / L to 20 g / L of a stabilizing agent as defined above, about 10 mM to 20 mM of a buffer solution as defined above.

  The subject of the invention is more particularly the above-mentioned use of a liquid composition as defined above, also referred to as MnP below, having a solid structure which is predominantly crystalline in the dry state after lyophilization, and comprising: 40 g / L of mannitol as a filler, 10 g / L of polyvinylpyrrolidone (PVP) as stabilizer, and 10 mM of a solution of Tris HCl (pH 7.8).

  The invention relates more particularly to the above-mentioned use of a liquid composition as defined above, also referred to as GP below, having a solid structure which is predominantly crystalline in the dry state after lyophilization, and comprising: - 40 g Of glycine as a filler, 10 g / L of polyvinylpyrrolidone as a stabilizer, and 10 mM of a solution of Tris HCl (pH 7.8).

  Advantageously, the liquid compositions as defined above, having an amorphous solid structure in the dry state after lyophilization, comprise: approximately 40 g / l to 60 g / l of a filler as defined above, about 10 g / L to 20 g / L of a stabilizing agent as defined above, about 10 mM to 20 mM of a buffer solution as defined above, and about 0.1 g / L L at 0.4 g / L of a nonionic surfactant agent as defined above.

  The invention relates more particularly to the above-mentioned use of a liquid composition as defined above, also referred to as MdSW below, having an amorphous solid structure in the dry state after lyophilization, and comprising: - 40 g / L maltodextrin as a filler, - 10 g / L sucrose as stabilizer, - 0.2 g / L Tween 80 as surfactant, - and 10 mM solution Tris HC1 (pH 7.8).

  The subject of the invention is more particularly the above-mentioned use of liquid compositions as defined above, characterized in that the proteins determined to be freeze-dried are proteins of pharmaceutical or analytical interest, in particular proteins of about 2000 to 3000 amino acids (or about 300 kDa molar mass), at a concentration of 3 g / mL to 10 g / mL (ie between 0.0003% and 0.001%) in the liquid composition.

  The invention relates more particularly to the aforementioned use of a liquid composition as defined above, characterized in that the stability of the lyophilized proteins is such that the activity of the proteins after storage in the freeze-dried state of 6 at 25 ° C is at least 80% of the activity of these same proteins before lyophilization.

  If necessary, the activity of the proteins after rehydration of the lyophilizate with osmosis water and storage in the liquid state of 3 months at 4 ° C. is at least 50% of the activity of these same proteins before lyophilization; by way of illustration, this threshold of 50% is reached in the case of the rehydration of the lyophilizate of toxin A, when the latter has been lyophilized in the aforementioned MnP, GP and MdSW compositions, and in the case of the rehydration of lyophilizate of toxin B, when the latter has been lyophilized in the MnP compositions and

  GP (see detailed description below).

  The subject of the invention is also a method of freeze-drying determined proteins, characterized in that it comprises: a step of dilution of the proteins in a composition as defined above, in proportions such that the proteins represent approximately 0 0003% to 0.001% dry matter in the liquid composition (for example 0.0004% toxin A and 0.0005% toxin B), - a step of freezing the solution obtained in the previous step at a temperature between - 35 ° C. and -45 ° C., in particular about -38 ° C., at a rate of between 0.3 ° C./min and 1 ° C./min, a step of primary drying of the frozen solution obtained in the preceding step by reduction of the pressure (in particular at a pressure of approximately 10 Pa at 20 Pa) and increasing the temperature of said frozen solution to a limit value of less than -18 ° C; a secondary drying step by increasing the temperature; e product 20 to about 20 C to 26 C, and maintain the product at this temperature for a time between 6h and 10h.

  Advantageously, the aforementioned freeze-drying method according to the invention is characterized in that the duration of the primary drying step is reduced by a factor of between 1.5 and 2, compared to the implementation of a freeze-drying process. conventional method not using the liquid compositions defined above for the stabilization of previously diluted proteins in said liquid compositions.

  The invention also relates to the application of the lyophilization process as defined above, to the stabilization of proteins in the dry state after lyophilization, in particular under the conditions described above.

  The subject of the invention is also a process for obtaining stable proteins in solution, in particular under the conditions described above, characterized in that it comprises a step of rehydration of the lyophilizate, obtained by carrying out the freeze-drying process as defined above, with osmosis water.

  The invention also relates to a method for screening liquid compositions as defined above, for carrying out the freeze-drying method on 5-determinate proteins, said liquid compositions being chosen from those for which: After storage in the freeze-dried state for 6 months at 25 ° C., the protein is at least 80% of the activity of these same proteins before freeze-drying, if necessary, the activity of the proteins after rehydration of the lyophilizate with a protein. reverse osmosis water and storage in the liquid state of 3 months at 4 C is at least 50% of the activity of 1 o these same proteins before lyophilization.

  the collapse temperature (Tcoll) of the liquid compositions is between -16 ° C. and -10 ° C.

  The subject of the invention is also a liquid composition, having a crystalline or amorphous solid structure in the dry state after freeze-drying, and comprising: approximately 40 g / l to 60 g / l of a charging agent having a temperature of collapse between -18 C and 0 C, - about 10 g / l to 20 g / l of a stabilizing agent, - about 10 mM to 20 mM of a buffer solution, said buffer solution does not crystallize during freezing of said composition, and, if appropriate, about 0.1 g / L to 0.4 g / L of a nonionic surfactant, said liquid composition being such that: the ratio by weight of the agent of load on the stabilizing agent is greater than 3: 1, when said composition has a predominantly crystalline structure after lyophilization, * the mass ratio of the stabilizing agent on the protein is greater than 1: 1, * it does not contain excipient of animal origin, * it has a collapse temperature of between -16 C and -10 C.

  The invention relates more particularly to a liquid composition as defined above, characterized in that the filler is chosen from: - polyols, such as mannitol or inositol, preferably mannitol, or the acids Amino acids, such as glycine or alanine, preferably glycine, when said compositions have a predominantly crystalline structure after lyophilization, or polysaccharides, especially those having an equivalent dextrose (DE) of less than 10, such as maltodextrin. , dextran or hydroxyethyl starch, preferably maltodextrin, when said compositions have an amorphous solid structure after lyophilization.

  The invention more particularly relates to a liquid composition as defined above, characterized in that the stabilizing agent is chosen from: - polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA), preferably PPV, when said compositions have a predominantly crystalline structure after lyophilization, or sucrose, trehalose, maltose or lactose, preferably sucrose, when said compositions exhibit an amorphous solid structure after lyophilization.

  The invention relates more particularly to a liquid composition as defined above, characterized in that the surfactant is selected from Tween 80, Tween 20, Triton X-100 or Brij 35, preferably Tween 80.

  The invention more particularly relates to a liquid composition as defined above, characterized in that the buffer solution is a solution of Tris HCl (pH 7.8), or potassium phosphate, preferably a solution of Tris -HC1.

  The invention more particularly relates to a liquid composition as defined above, having a mainly crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of mannitol as filler, - g / L of polyvinylpyrrolidone as a stabilizing agent, - 10 mM of a Tris HCl solution (pH 7.8).

  The invention more particularly relates to a liquid composition as defined above, having a predominantly crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of glycine as a filler, 10 g / l of polyvinylpyrrolidone as stabilizer, 10 mM of a solution of Tris HCl (pH 7.8).

  The invention more particularly relates to a liquid composition as defined above, having an amorphous solid structure in the dry state after lyophilization, and comprising: - 40 g / l of maltodextrin as a filler, 10 g / L in the liquid sucrose composition as a stabilizing agent, 0.2 g / L Tween 80 as surfactant, 10 mM Tris HCl solution (pH 7.8).

  The invention also relates to a liquid composition as defined above, characterized in that it comprises a specific protein chosen from proteins of pharmaceutical interest, or analytical, in particular proteins of approximately 2000 to 3000 amino acids. (or molar mass of about 300 kDa), at a concentration of about 3 g / mL to 10 g / mL (ie 0.0003% and 0.001%) in the liquid composition.

  The present invention will be further described with the aid of the description which follows of the liquid compositions as defined above, of their use in the context of the implementation of the lyophilization of toxins A and B of Clostridium difficile, and of the best stability of these lyophilized proteins compared to the same lyophilized proteins by conventional techniques in this field.

  1. Procedure 1) Mixing rules a) Due to health safety constraints, no excipients of animal origin have been used. Thus bovine serum albumin (BSA), often proposed in the literature as a bulking agent and stabilizer, has been ruled out.

  b) After the selection of 8 molecules, a preliminary screening of the liquid compositions was performed according to their physical properties. 29 liquid compositions (see Table I) were finally established according to the following rule: All mixtures contain at least: - a filler (and / or stabilizer) present at a concentration between 40 g / L and 60 g / L and conferring a mechanical strength to the porous structure during the departure of the ice and thus avoiding the entrainment of the protein with water vapor during the primary drying step, and a buffer agent does not crystallize during the freezing step, a Tris-HCl buffer present at a concentration of 10 mM. Table I. Composition of the 29 Liquid Compositions Tested (a) Bulking Agent Bulking Agent and / or Stabilizer Cryoprotective Surfactant (Crystalline) Stabilizer (Amorphous Solid) (Amorphous Solid) (b) Glycine Mannitol PVP Maltodextrin Sucrose Maltose PEG Tween 80 (25 KDa) (5-8 DE) G Mn P Md SMEW Md 40 g / L MdW 40 g / L 0. 02% MdSW 40 g / L 10 g / L 0.02% MdS 40 g / L 10 g / L MgE 40g / L L 10g / L MgE 40g / L 10g / L 10g / LP 40g / L PE 40g / L 10g / L PW 40g / L 0.02% PS 40g / L 10g / L PM 40g / L L 10g / L PSW 40g / L 10g / L 0.02% G 40g / L GM 40g / IOL g / L GS 40g / L 10g / L GSW 40g / L 10g / L 0.02% GSE 40g / L 10g / L 10g / L GE 40g / L 10g / L GW 40g / L 0.02% GMd 40g / L 10g / L GP 40g / L 10g / L GPW 40g / L 10 g / L 0.02% GPE 40 g / L 10 g / L 10 g / L Mn 40 g / L MnP 40 g / L 10 g / L MnS 40 g / L 10 g / L MnW 40 g / L 0.02% MnPW 40 g / L 10 g / L 0.02% S 40 g / L (a) Criterion for definition of the 29 formulas: All the mixtures contain at least: a loading agent and / or stabilizer, and a buffer consisting of a solution mM tris-HCl (pH 7.8), or a formula at a final rate of 4% to 6% dry matter.

  The formulas have a final concentration of toxin A and toxin B of 5.14 g / ml and 3.88 mg / ml, respectively.

  (b): These bulking agents can also act as a stabilizer and, in this case, if this molecule is associated with another filler, it is added only at 10 g / l.

  2) Measurement of the Physical Properties of the Liquid Compositions and Evaluation of the Stability of the Lyophilized Proteins with these Compositions a) In order to select the liquid composition meeting the compromise of protein stability and good productivity of the lyophilization process, the physical properties (transition temperatures vitreous and collapse) were measured.

  b) The stability of the proteins was evaluated after lyophilization, after 6 months of storage at 25 C in the freeze-dried state and after 3 months of storage at 4 C in the liquid state after rehydration. The method of monitoring the activity of proteins is specific to the proteins studied.

  l0 II. Materials and Methods 1) Proteins of the study Two model proteins were studied: the toxins A and B produced by Clostridium difficile. These proteins are used in diagnostic kits and must be rehydrated before use. Toxins A and B are large proteins (from 260 to 308 KDa). These proteins were obtained in the industrial laboratory and introduced into the liquid compositions at a final concentration of toxin A and toxin B of 5.14 g / ml and 3.88 g / ml respectively (Table I).

  2) Differential Enthalpy Analysis The thermal analysis of the solutions and lyophilized products was conducted using a differential scanning calorimeter with power compensation (DSC, Pyris model 1, Perkin Elmer LLC, Norwalllk, CT, USA) equipped with a source of liquid nitrogen (CryoFill, Perkin Elmer). Equipment calibration was performed using cyclohexane, mercury and gallium. 15 L of liquid sample, or 10 mg of freeze-dried sample was placed in an aluminum capsule and an empty capsule was used as a reference. The cooling and heating rates were set at 10 ° C.min -1. The glass transition temperature is defined as the median value of the specific heat change associated with the glass transition event.

  The liquid samples were cooled to -120 ° C. and then heated to 25 ° C. This thermal cycle was repeated. For certain liquid samples comprising a crystalline filler, specific thermal cycles were applied to promote complete crystallization of the filler (mannitol or glycine).

  The freeze-dried samples were cooled to -40 ° C and heated to 200 ° C. To eliminate any signal interference with enthalpic relaxation phenomena, specific thermal cycles were applied such as annealing. , or a sequence repetition of short heating and holding steps (Perkin Elmer Stepscan DSC software).

  3) Cryo-microscopy The collapse temperature (collapse temperature, Tcoll) was measured using a cryo-desiccation cell (Linkam Scientific Instruments, Surrey, UK) equipped with a nitrogen cooling system and a temperature programmer. A 5 L sample was placed on a 16 mm diameter quartz slide and covered with a 13 mm diameter quartz slide. A metal template, placed between the two blades, ensures a constant thickness of the sample. Silicon oil was deposited between the sample and the temperature control plate. The direct observations of the freeze-dried structure were obtained by means of a Nikon Elipse E600 microscope (Nikon, Japan) The determined collapse temperature (Tcoll) corresponds to the minimum temperature from which, during freeze-drying, there is a loss of the structure established during freezing [5].

  The measurement protocol is described in an earlier publication [5]. The sample was frozen at 10 ° C. at a temperature slightly lower than its Tg.sup.- After lowering the pressure in the cell (approximately 1 Pa), the temperature of the sample was maintained. for 10 to 20 minutes until a dry region is obtained which is wide enough to detect a deterioration of the structure, then the temperature has been gradually increased in steps of 5 C or 1 C when the onset of collapse has For compositions comprising a crystalline filler (mannitol or glycine), a holding step at -20 C for 20 min was applied after the freezing step to allow complete crystallization of the filler.

  4) Lyophilization cycle The liquid compositions were distributed in 4 mL glass flasks (with a final volume of 1 mL). The flasks previously placed in a tray were placed on the shelf of a freeze dryer SMH 15 (Usifroid, Maurepas, France). The following steps were applied: (1) cooling the shelf to -60 C at a rate of 1 C / min and maintaining the shelf at -60 C for 2 hours; (2) decrease the pressure up to 10 Pa, increase the shelf temperature to -25 C and maintain the shelf at -25 C for 50h; (3) increase the shelf temperature up to 25 C and maintain the temperature for 8h. At the end of the cycle, the vacuum was broken by injecting a stream of dry nitrogen gas, the flasks were quickly capped and packaged under vacuum in aluminum bags.

  Thermocouples (types K) were placed at the bottom of two bottles in order to follow the temperature of the product. During sublimation (primary desiccation), the temperature of the product was maintained at about -34 C.

  5) Measurement of antigenic activity The antigenic activity of toxins A and B was measured by a quantitative ELISA test, developed by bioMérieux. The activity was measured immediately after lyophilization, after storage for 6 months at 25 ° C. in the freeze-dried state and after storage for 3 months at 4 ° C. in the liquid state after rehydration. The coefficient of variation of this assay method is 15%.

  III. Results 1) Physical properties (see Table II) According to the physical behavior of the filler during freezing, the liquid compositions are classified into two categories: compositions comprising a filler agent present in the state amorphous solid after freezing: they are characterized by a value of the glass transition temperature of the cryo-concentrated phase (Tg ') close to the collapse temperature (Tcoll). The maltodextrin compositions have Tcoll values 10 C higher than those of the polyvinylpyrrolidone (PVP) compositions.

  the compositions comprising a filler present in the crystalline state after freezing: they are characterized by Tcoll values which are much higher (from 10 ° C. to 20 ° C.) than the Tg 'values.

  The Tcoll collapse temperature, representative of the loss of the freeze-dried structure appears as a more relevant criterion to consider for the optimization of the freeze-drying process.

  Table II. Synthesis of the physical properties of the liquid compositions studied DSC Cryo-microscopy Tg '(C) Te (C) Tcoll (C) Tmelt (C) Amorphous solid (Tg' close to Tcoll) Md -13 -10 MdW -14 -9 MdSW -15 - 14 MdS -17 -14 MdE -10 -18 -15 MdSE -16 -21 -18 P -27 -25 -23 PE -23 -22 -26 PW -26 -26 PM -24 -26 PS -28 -27 PSW -28 -27 S -36 -35 Crystalline Solid ('lg' Tcoll) G * -4 -16 -5 / -4 GM * -37 -4 -15 -5 / -4 GS * -38 -4 -15 -5 / -4 GSW * -39 -4 -15 -5 / -4 GSE * -38 -19; -4 -18 -5 / -4 GE * -17; -4 -16 -5 / -4 GW * -4 -20 -5 / -4 Gl ^ / Id * -20 -4 -10 -5 / -4 GP * -28 -4 -15 -5 / -4 GPW * -27 - 4 -15 -5 / -4 GPE * -18; -4 -18 -5 / -4 Mn + -32; -25 -2 -10 -3 / -2 MrP * -25 -2 -14 -3 / -2 MES * -38 -2 -20 -3 / -2 MIiW * -32; -25 -2 -10 -3 / -2 MnPW * -26 -2 -3 / -2 (a) measured by DSC, average value of at least two measurements (b) measured by cryomicroscopy, average value of at least two measurements Tg ': Glass transition temperature of the cryoconcentrated phase Tcoll: Collapse temperature (collapse) Te: Eutectic temperature Tmelt: Melting temperature of the solution *: Application of a specific thermal cycle allowing complete crystallization of the silver The protein activity (see Table III) immediately after lyophilization, the antigenic activity of the two proteins represents more than 80% of the antigenic activity before lyophilization for all the compositions. tested liquids. No loss of antigenic activity was therefore observed.

  5. For the selection of the most efficient liquid compositions, the following stability criteria were retained: an antigenic activity greater than 80% of the value before lyophilization after storage for 6 months at 25 ° C. in the freeze-dried state and or an antigenic activity of at least 50% after storage for 3 months at 4 C in the liquid state.

  The liquid compositions MnP and GP, including 4% of a crystalline filler (mannitol or glycine, respectively) and 1% of a stabilizing agent, polyvinylpyrrolidone, ensure the stability of the two proteins both in the freeze-dried state. in the liquid state. The addition of a non-ionic surfactant agent, Tween 80 (denoted W), does not significantly improve the stability of the two proteins (cf MnPW and GPW with respect to MnP and GP, respectively).

  The liquid MdSW composition, including 4% maltodextrin, 1% sucrose and 0.02% Tween 80, appears very effective for the stabilization of the two proteins in the freeze-dried state.

  In addition, these three liquid compositions (MnP, GP and MdSW) have high Tcoll collapse temperature values (-15 C to -14 C), thus making it possible to shorten the lyophilization cycle.

  Table III. Antigenic activity of freeze-dried proteins after storage for 6 months at 25 C in the dry state and after storage for 3 months at 4 C in the liquid state after rehydration Toxin A Toxin BT colt (C) Dry Liquid Dry Liquid 6 months at 25 C 3 months at 4 C 6 months at 25 C 3 months at 4 C Amorphous solid Md -10 54 0 68 6 MdW -9 81 5 89 5 MdSW -14 86 50 89 8 MdS -14 61 0 78 3 MdE - 15 83 96 77 12 MdSE -18 88 77 65 15 P -25 72 68 94 82 PE -26 89 85 73 62 PW -26 81 67 103 96 PM -26 90 95 75 44 PS -27 85 79 87 83 PSW -27 96 81 111 110 S -35 94 7 90 14 Crystalline solid G -16 17 59 55 97 GM -15 71 100 75 86 GS -15 84 72 80 27 GSW -15 74 86 101 139 GSE -18 72 84 77 110 GE - 16 30 18 61 90 GW -20 7 85 48 93 GMd -10 67 6 83 11 GP -15 87 72 96 48 GPW -15 90 85 92 55 GPE -18 77 83 91 95 Mn -10 21 95 43 51 MnP -14 95 84 89 70 MnS -20 84 46 78 63 MnW -10 11 83 60 122 MnPW - 15 99 43 102 13 (a) The unit in which the activity of the protein is expressed is the percentage calculated by reference. This is due to antigenic activity before lyophilization, since the activity immediately after lyophilization is greater than 80%. The value given is the average of two measurements. The criterion of stability is an activity greater than or equal to 80.

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Claims (1)

19 CLAIMS   1. Use of a liquid composition in the context of the implementation of the lyophilization process on predetermined proteins previously diluted in said composition, with a view to the stabilization of these proteins in the dry state after lyophilization, and, where appropriate optionally in the liquid state after rehydration of the lyophilizate obtained, said composition having a predominantly crystalline or amorphous solid structure in the dry state after lyophilization, and comprising: -about 40 g / l to 60 g / l of a charging agent having a collapse temperature between -18 C and 0 C, - about 10 g / l to 20 g / l in the liquid composition of a stabilizing agent, - about 10 mM to 20 mM of a buffer solution, said solution buffer not crystallizing during the freezing of said composition, and, if appropriate, about 0.1 g / l to 0.4 g / l of a nonionic surfactant, said liquid composition being such that: ratio in mass of the agent of load on the stabilizing agent is greater than 3: 1, when said composition has a predominantly crystalline structure after lyophilization * the ratio by weight of the stabilizing agent on the protein is greater than 1: 1, * it does not contain excipient of animal origin, * it has a collapse temperature between -16 C and -10 C.   2. Use according to claim 1, characterized in that the filler is chosen from: polyols, such as mannitol or inositol, preferably mannitol or amino acids, such as glycine or alanine, preferably glycine, when said compositions have a predominantly crystalline structure after lyophilization, or polysaccharides, especially those having an equivalent dextrose (DE) of less than 10, such as maltodextrin, dextran, or hydroxyethyl starch, preferably, maltodextrin, when said compositions exhibit an amorphous solid structure after lyophilization.   3. Use according to claim 1 or 2, characterized in that the stabilizing agent is chosen from: polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA), preferably PVP, when said compositions have a structure predominantly crystalline after lyophilization, or sucrose, trehalose, maltose or lactose, preferably sucrose, when said compositions have an amorphous solid structure after lyophilization.   4. Use according to one of claims 1 to 3, characterized in that the surfactant is selected from Tween 80, Tween 20, Triton X-100, or Brij 35, preferably Tween 80.   5. Use according to one of claims 1 to 4, characterized in that the buffer solution is a solution of Tris HCl (pH 7.8), or potassium phosphate, preferably a solution of Tris-HCl.   6. Use according to one of claims 1 to 5, a liquid composition, having a mainly crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of mannitol as a filler, 10 g / l of polyvinylpyrrolidone as a stabilizing agent, 10 mM of a solution of Tris HCl (pH 7.8).   7. Use according to one of claims 1 to 5, a liquid composition, having a mainly crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of glycine as a filler, 10 g / l of polyvinylpyrrolidone as a stabilizing agent, 10 mM of a solution of Tris HCl (pH 7.8).   8. Use according to one of claims 1 to 5, a liquid composition, having an amorphous solid structure in the dry state after lyophilization, and comprising: - 40 g / l of maltodextrin as a filler, 10 g / L of sucrose as a stabilizing agent, 0.2 g / L of Tween 80 as surfactant, 10 mM of a solution of Tris HCl (pH 7.8).   9. Use according to one of claims 1 to 8, characterized in that the determined proteins are proteins of pharmaceutical interest, or analytic, including proteins of about 2000 to 3000 amino acids (or molecular weight of about 300 kDa), representing approximately between 3 g / mL and 10 g / mL (ie 0.0003% and 0.001%) of dry matter in the liquid composition.   10. A process for lyophilization of determined proteins, characterized in that it comprises: a step of diluting the proteins in a composition as defined in one of claims 1 to 8, in proportions such that the proteins represent approximately 0. 0003% to 0.001% of dry matter in the liquid composition, - a step of freezing the solution obtained in the preceding step at a product temperature of between -35 ° C. and -45 ° C., in particular about 38 C, at a speed between 0.3 C / min and 1 C / min, - a primary drying step of the frozen solution obtained in the previous step by decreasing the pressure and increasing the temperature of said frozen solution until at a lower temperature below -18 ° C., a secondary drying step by increasing the temperature of the product to about 20 ° C. to 26 ° C. for a time of between 6:00 and 10:00.   11. Application of the lyophilization process according to claim 10, for the stabilization of proteins in the dry state after lyophilization.   12. Process for obtaining proteins in stabilized form in solution, characterized in that it comprises a step of rehydrating the lyophilizate obtained by carrying out the freeze-drying method according to claim 10.   13. A method of screening liquid compositions as defined in one of claims 1 to 5, for the implementation of freeze-drying methods of determined proteins, said liquid compositions being chosen from those for which: the activity of the proteins after freeze-dried storage for 6 months at 25 ° C is at least 80% of the activity of these same proteins before lyophilization, - where appropriate, the activity of the proteins after rehydration of the lyophilizate and a storage at the liquid state of 3 months at 4 ° C. is at least 50% of the activity of these same proteins before lyophilization.   the collapse temperature of the liquid compositions is between -16 ° C. and -10 ° C.   14. A liquid composition, having a predominantly crystalline structure or amorphous solid in the dry state after lyophilization, and comprising: - about 40 g / l to 60 g / l of a filler having a collapse temperature of -18 C and 0 C, - about 10 g / L to 20 g / L of a stabilizing agent, - about 10 mM to 20 mM of a buffer solution, said buffer solution does not crystallize upon freezing of said composition, and, where appropriate, about 0.1 g / L to 0.4 g / L of a nonionic surfactant, said liquid composition being such that: the mass ratio of the filler to the agent stabilizer is greater than 3: 1, 15 when said composition has a predominantly crystalline structure after lyophilization * the ratio by weight of the stabilizing agent on the protein is much greater than * it does not contain excipient of animal origin, 20 * It has a collapse temperature between -16 C and -10 C.   15. Liquid composition according to claim 14, characterized in that the filler is chosen from: polyols, such as mannitol or inositol, preferably mannitol, or amino acids, such as glycine or alanine, preferably glycine when said compositions have a predominantly crystalline structure after lyophilization, or polysaccharides, especially those having an equivalent dextrose (DE) of less than 10, such as maltodextrin, dextran, or starch hydroxyethyl, preferably maltodextrin, when said compositions exhibit an amorphous solid structure after lyophilization.   16. A liquid composition according to claim 14 or 15, characterized in that the stabilizing agent is chosen from: polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA), preferably PVP, when said compositions have a structure predominantly crystalline after lyophilization, or sucrose, trehalose, maltose or lactose, preferably sucrose, when said compositions have an amorphous solid structure after lyophilization.   17. Liquid composition according to one of claims 14 to 16, characterized in that the surfactant is selected from Tween 80, Tween 20, Triton X-100, or BRIJ 35, preferably Tween 80.   18. Liquid composition according to one of claims 14 to 17, characterized in that the buffer solution is a solution of Tris HCl (pH 7.8), or potassium phosphate, preferably a solution of Tris-HCl.   19. Liquid composition according to one of claims 14 to 18, having a mainly crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of mannitol as a filler, - 10 g / l. Polyvinylpyrrolidone as stabilizer, 10mM Tris HCl solution (pH 7.8).   20. Liquid composition according to one of claims 14 to 18, having a substantially crystalline structure in the dry state after lyophilization, and comprising: - 40 g / l of glycine as a filler, - 10 g / l of polyvinylpyrrolidone as a stabilizing agent, 10mM Tris HCl solution (pH 7.8).   21. Liquid composition according to one of claims 14 to 18, having an amorphous solid structure in the dry state after lyophilization, and comprising: - 40 g / l of maltodextrin as a filler, -10 g / Sucrose as stabilizer, 0.2 g / L Tween 80 as surfactant, 10 mM Tris HCl solution (pH 7.8).   22. Liquid composition according to one of claims 14 to 21, characterized in that it comprises a specific protein selected from proteins of pharmaceutical interest, or analytical, including proteins of about 2000 to 3000 amino acids (or molar mass of approximately 300 kDa), representing approximately between 3 g / mL and 10 g / mL (ie 0.0003% and 0.001%) of dry matter in the liquid composition.