MXPA98009774A - Improved process to stabilize protei - Google Patents

Abstract

The present invention relates to an improved process for preventing the formation of protein aggregates in a reconstituted lyophilizate of a pharmaceutical composition of a protein, in which a buffered aqueous solution of the protein is frozen, thawed, divided into compartments of Injectable amounts and said compartments are lyophilized and which is characterized in that the buffered aqueous solution of the protein contains potassium phosphate buffer as buffer substance and the ratio of potassium ions to sodium ions in the solution is at least 10: 1, can advantageously used to produce stable lyophilisates of pharmaceutical compositions

Description

IMPROVED PROCESS TO STABILIZE PROTE NAS Field of the Invention The invention relates to an improved process for the stabilization of proteins in a freezing or lyophilization process or during storage at low temperatures.

BRIEF DESCRIPTION OF THE INVENTION Proteins such as enzymes or antibodies as well as fragments thereof, are unstable and susceptible to losing their activity and / or form soluble or insoluble aggregates in aqueous solutions and when stored at low temperatures (below 0 ° C) and in particular in repeated freezes and thaws these aggregates are made visible forming particles and thereby turbidity. However, this formation of aggregates and / or particles can not be tolerated, except in trace amounts, for protein pharmaceutical compositions. A pharmaceutical composition must be a clear solution and if it is present as lyophilized, it must be FEF. 028857 also form a transparent solution free of particles when it is reconstituted, which must also be free of soluble protein aggregates. Numerous processes and additives are known for stabilizing proteins in solutions. For example, the stabilization of heat shock proteins such as HSP25 is described for example in EP-A 0 599 344. The stabilization of antibodies by the addition of block polymers composed of polyoxylene and polyoxyethylene and by phospholipids is described in EP-A 0 318 081. EP-A 0 025 275 discloses the stabilization of immunoglobulins by the addition of a salt of a basic nitrogen-containing substance such as arginine, guanidine or imidazole. Other suitable additives for stabilization are polyethers (EP-A 0 018 609), glycerin, albumin and dextran sulfate (US Patent 4,808,705), detergents such as Tween's 20 m (DE 26 52 636, GB 8514349), "chaperones" "such as GroEL (Mendoza, JA Biotechnol, Tech. 10 (1991) 535-540), citrate buffer (WO 93/22335) or chelating agents (WO 91/15509). Although these additives make it possible to stabilize the proteins to a certain degree in aqueous solution, it has been found, however, that none of the methods known in the prior art is suitable for the stabilization of proteins when repeated freezing and thawing processes take place. that no soluble or insoluble aggregate is formed or only significant amounts for therapeutic purposes during thawing, during storage at temperatures below 0 ° C, or when a solution is reconstituted after lyophilization. In EP-A 0 314 095 a lyophilisate of a plasma protein such as factor VIII is described, which contains histidine buffer as a buffer substance and calcium chloride as an additive and is present with a high ionic strength (0.35 a 1.2 moles / liter of NaCl). A lyophilisate of a plasma protein such as factor VIII is described in EP-A 0 315 968, which contains from 0.5 to 15 mmoles / liter of sodium chloride or potassium chloride, 0.01 to 10 mmoles / liter of lysine hydrochloride and 0.2 to 5 mmoles / liter of histidine as buffer ion. However, histidine buffer is not suitable for stabilizing the protein or preventing the formation of aggregates and particles when the lyophilized proteins are reconstituted.

Accordingly, the object of the invention is to provide a process that can substantially prevent the formation of aggregates and particles when the lyophilisates of pharmaceutical protein compositions are reconstituted. Therefore, the invention relates to an improved method for preventing the formation of protein aggregates in a solution of a pharmaceutical composition of a protein, preferably an antibody, which is reconstituted from a lyophilized, wherein a buffered aqueous solution of the protein is frozen, thawed, divided into compartments of injectable amounts and these compartments are lyophilized, which is characterized in that the buffered aqueous solution of the protein contains potassium phosphate buffer as buffer substance and the ratio of potassium ions to sodium ions in the solution is 10: 1 or greater. The aqueous buffer solution preferably does not contain, essentially, any sodium ion. The invention allows protein pharmaceutical compositions, in particular, proteins having a tendency to dimerize or polymerize such as antibodies, which are formulated in a stable pharmaceutical composition, with a neutral pH (on the order of 6-8, preferably pH 6.5 - 7.5). Proteins such as antibodies tend to aggregate in an order of neutral pH if the solution is frozen (optionally in lyophilized form) once or several times and thawed again. A pharmaceutical composition is especially advantageous in potassium phosphate buffer in an order of pH between 6 and 8, at a buffer concentration between 10 and 300 mmoles / liter, preferably between 50 and 250 mmoles / liter, in which it is present the lowest possible number of sodium ions in the pharmaceutical composition. An adequate ratio of potassium ions to sodium ions in the solution is 10: 1 or greater. It is particularly preferable that the potassium phosphate buffer is used only as a buffer substance in the pharmaceutical composition and no sodium salt (such as for example sodium chloride) is added. In this case almost no sodium ion is present in the pharmaceutical composition or only contains them in such small amounts that they do not cause formation of protein aggregates during repeated freezing or thawing. It has been found that lyophilisates of protein solutions that have been frozen at least once during the production process can then be substantially reconstituted without formation of turbidity if the potassium phosphate buffer is used as the buffer substance. Customary buffers such as sodium phosphate buffer, histidine buffer or citrate buffer lead to the formation of aggregates in said process, which are mainly composed of the protein and thus also lead to turbidities to a considerable degree. The frozen protein solutions are already completely frozen at -15 ° C, have eutectic points above about -15 ° C and thus can already be stored at this temperature or at lower temperatures, preferably for example at -20 ° C. Since a solution is only completely frozen below the eutectic temperature, this means that a protein in a phosphate buffer containing sodium ions is subjected to a great stress during frozen storage (usually at -20 ° C) and during the frozen / thawed process when compared to a buffer free of sodium ions or in a buffer in which the concentration of sodium ion is very low. According to the invention this effort is avoided in the formulations mentioned above, in which the elimination of the formation of aggregates and particles is obtained as a result. This formulation allows stable storage of the protein solution at -20 ° C, which allows cost savings. The potassium phosphate buffers in contrast to the sodium phosphate buffers present only a slight variation of the pH (preferably at most +1 pH unit, preferably particularly at most +0.5 pH units) during the process of freezing. It has been found that the concentration of the phosphate buffer should be at least 10 mmole / liter, preferably about 50 mmole 1 / liter or greater, in order to effectively prevent the formation of particles. Since the osmolarity should not be too high (it must be advantageously of the physiological order, preferably about 300 Osm once reconstituted (+ 20 mOsm, a range of 100 to 500 mOsm is also suitable)) in pharmaceutical compositions (ie, preferably in the reconstituted solution), the concentration of the buffer substance or optionally the sum of the buffer substance and the salt should not be greater than 250-300 mmole / liter. The concentration of the buffer is preferably between 50 and 250 mmole / liter in the compartment. However, higher concentrations of buffer substance and / or salt can be tolerated in the production of solutions (bulk material) used to produce the compartments. If a salt additive is desired in the pharmaceutical composition especially for adjusting the ionic strength, it is advantageous according to the invention, neither to use sodium salts nor to choose a concentration of sodium ions so that it is substantially lower than the concentration of sodium ion. potassium ions. It is therefore convenient to add a potassium salt such as potassium chloride instead of the usual sodium chloride. However, it has been found that low amounts of sodium salts (for example about 10 mmol / l or less) do not interfere, provided that the ratio of potassium ions to sodium ions is 10: 1 or greater. It is not possible to add calcium salts such as, for example, calcium chloride, since calcium phosphate precipitates with such addition and, therefore, apart from the formation of undesirable turbidity, the buffering effect of potassium phosphate according to the invention , it is canceled. The insoluble aggregates whose formation must be prevented in the process according to the invention, are understood to be essentially protein aggregates whose size is usually at least 1 μm, but may also be of an order greater than 10 μm. The particles can be determined by suitable particle counting methods using particle counting instruments, which can be purchased commercially, such as, for example, the AccuSizer 700 instrument from PSS (Particle Sizing Systems, USA). According to the invention an improvement of the process is achieved, if the number of particles between 2 and 400 μm / ml is <; 3000 or the number of particles between 10 and 400 μm / ml is 2000 or less. According to the USP (US Pharmacopoeia), a maximum of 6000 particles is allowed in a range above 10 μm, and a maximum of 600 particles in a range above 25 μm, per injected dose of a pharmaceutical preparation. This can be achieved according to the invention, in a simple manner, for therapeutic protein compositions. Proteins (Polypeptides) are included within the scope of the invention as proteins or fragments and proteins, as well as chemically modified proteins. The proteins to be stabilized for pharmaceutical compositions are preferably antibodies, antibody fusion proteins such as immunotoxins, enzymes and protein hormones such as erythropoietin, somatostatin, insulin, cytokines, interferons or plasminogen activators. The compartments, within the scope of the invention, are understood as aliquots of the protein solution, which, optionally, after further processes (addition of other pharmaceutically acceptable substances), are suitable as pharmaceutical compositions, preferably for administration as an injectable. to the patients. The pH range in which the pharmaceutical composition is stabilized by the potassium phosphate buffer is preferably a slightly acidic, neutral or slightly alkaline margin (about pH 6-8, preferably around pH 7). According to the invention it is preferable to add a nonionic detergent such as a polysorbate (for example Tween ^ dO), preferably at a concentration of at most 0.1% by weight and at least 0.01% by weight. In addition, it is preferable to add cryoprotectants or ice makers such as a non-reducing sugar (preferably sucrose or trehalose), advantageously at a concentration of at least 10 mg / ml, preferably about 30-100 mg / ml. Accordingly, another object of the invention consists of a storage form of protein with low content of aggregates, fusible solid, essentially amorphous, composed of a frozen solution of the protein and potassium phosphate buffer as the main buffer substance, in the which the ratio of potassium ions to sodium ions in the solution is at least 10: 1. Regardless of the concentration of potassium ions and the residual content of sodium ions, the ratio of potassium ions to sodium ions should be at least 10: 1, preferably at least 50: 1. It is particularly preferable to employ essentially a potassium buffer free of sodium ions. In another preferred embodiment of the invention, the pharmaceutical composition contains a protein that has been produced by an in vitro cell culture (e.g. the recombinant production or culture of a hybridoma cell line, to produce monoclonal antibodies). In this case it is convenient either to add a potassium salt and / or potassium phosphate buffer, with the first addition of salt and / or buffer, or to re-filter the last time in the isolation and purification process. This allows stable interim storage of the polypeptide preparation below 0 ° C. Hardening is understood as an exchange of ions for example by dialysis. In the process of purification and isolation of the protein the concentration of the buffer or of the salt can be effectively greater than 50-100 mmol / l before the compartmentalization since these compositions are not used therapeutically. However, it is essential that an osmolarity that has to be suitable for an injectable composition be adjusted before compartmentalization. The following examples, publications and figures clarify in more detail the invention, the protective scope of which results from the patent claims. The described methods should be understood as examples that describe in more detail the subject matter of the invention, even after modifications.

Brief description of the figures Figure 1 shows the determination of the eutectic points of several buffers and saline solutions.

Figure 2 shows the deviation of the pH value during freezing of the phosphate buffers. Figure 3 shows the formation of particles of solutions of an antibody (against L-selectin) in several solutions of buffers (A, B, C) after shearing or freezing / thawing. A: AB in 10 mmoles / liter KP, 150 mmoles / liter of NaCl, pH 7; B: AB in 100 mmoles / liter of KP, pH 7.2; C: AB in 100 mmoles / liter of KP, 0.01% by weight of Tween ^ dO, pH 7.2; a: centrifugation (starting material); b: after the shear stress (30 seconds of Vortex mixing), c: after six freeze / thaw cycles (-20 ° C). Figure 4 shows the formation of particles of solutions of an antibody against HBV in several buffer solutions (A, B and C) after shearing or freezing / thawing. A: AB in 10 mmoles / liter of KP, 30 mmoles / liter of NaCl, pH 6.5; B: AB in 100 mmoles / liter of KP, pH 7.2; C: AB in 100 mmoles / liter of KP, 0.01% by weight of Tween®80, pH 7.2; Figure 5 shows HPLC analysis of gel permeation of soluble aggregates in protein solutions (humanized IgH according to example 3) after storage at temperatures below 0 ° C. A: AB in 10 mmoles / liter of KP, 150 mmoles / liter of NaCl, pH 7.0; B: AB in 100 mmoles / liter of KP, pH 7.2. Example 1 Eutectic temperatures of various buffer solutions and saline solutions From Figure 1, it is evident that the eutectic temperature of buffers containing NaCl is about 10 ° C lower than that of NaCl-free buffers or solutions containing KCl in Place of NaCl. Since a solution is only completely frozen below the eutectic temperature, this means that a protein in a phosphate buffer containing NaCl is subjected to a higher stress than in a NaCl-free buffer during the congwelado storage (usually at 20 ° C ), and during the freeze / thaw process. According to the invention this effort is avoided in the formulations formulated above, which suppresses the formation of aggregates and particles. This formulation allows stable storage of the protein solution at -20 ° C, whereby cost savings can be achieved.

Example 2 Variation of pH value during freezing of phosphate buffers It is evident from Figure 2 that in phosphate buffers containing NaCl the pH value decreases to a high degree during the freezing process due to the disodium phosphate precipitated . The pH value remains largely constant in the NaCl-free potassium phosphate buffer.

Example 3 Formation of particles in the protein solutions after the shear stress or freeze / thaw. Solutions of a humanized IgG (anti-L selectin antibody) in several buffers (A, B, C) were analyzed to determine the particulate content (Accu Sizer, Particle Sizing Systems, USA): A) AB in 10 mmole / liter of KP, 150 mmoles / liter of NaCl, pH 7 B) AB in 100 mmoles / liter of KP, pH 7.2 C) AB in 100 mmoles / liter of KP, 0.01% by weight of Tween®80, pH 7 , 2 a) centrifugation (starting material) b) after the shearing stress (30 seconds of treatment in the Vortex) c) after six freeze / thaw cycles (-20 ° C).

The data of Figure 3 refer each to a sample of 0.7 ml. From Figure 3 it follows that the formation of particles is suppressed according to the invention by the use of e sodium-free potassium phosphate buffers. This effect can be increased by the addition of a non-ionic detergent (Tween * 80, 0.01% by weight).

Example 4 Formation of particles in protein solutions after shearing or freezing / thawing. Solutions of an anti HBV antibody were analyzed in several in several buffers (A, B, C) to determine the content of particles (Accu Sizer, Particle Sizing Systems): A) AB in 10 mmoles / liter of KP, 30 mmoles / liter of NaCl, pH 6.5 B) AB in 100 mmoles / liter of KP, pH 7.2 C) AB in 100 mmoles / liter of KP, 0, 01% by weight of Tweenf80, pH 7.2 a) centrifugation (starting material) b) after the shear stress (30 seconds of treatment in the Vortex) c) after six freeze / thaw cycles (-20 ° C) ) The data of figure 3 each relate a sample of 0.7 ml. It can be seen from FIG. 4 that the formation of particles is suppressed according to the invention by the use of sodium-free potassium phosphate buffers. This effect can be increased by the addition of a non-ionic detergent Example 5 Prevention of the formation of soluble aggregates during the storage of protein solutions (humanized IgG according to example 3) at temperatures below 0 ° C Protein solutions were stored for several weeks at -20 ° C in A) mM of potassium phosphate, 150 mM of NaCl, pH 7.0, and B) in 100 mM of potassium phosphate, pH 7.2. Analysis of soluble aggregates and the native protein were performed by gel permeation HPLC (Figure 5). According to the invention a considerably smaller number of protein aggregates are formed with the NaCl-free buffer than with the NaCl-containing buffer. This is due primarily to the fact that a variation of the pH value in the NaCl-free buffer has been substantially prevented and the storage temperature is considerably lower than the eutectic temperature (see also examples 1 and 2). Example 6: Formation of particles in protein solutions after the freeze / thaw stress The MAB L-selectin, MAB HBV antibodies were analyzed; MAB PDGF-R and MAB LNGF-R in several buffers, to determine the particle content before and after freezing / thawing (6 x freezing / thawing) (Accu Sizer, Particle Sizing Systerr. ^ 1 (see results in table 1, Cprot .: protein concentration.) Particles with a size of 2-400 μm per ml were detected). It is evident that the formation of particles is suppressed according to the invention by the use of sodium-free potassium phosphate (KP) buffers. This effect can be increased by the addition of a non-ionic detergent.

Table 1 List of references DE 26 52 636 EP-A 0 018 609 EP-A 0 025 275 EP-A 0 314 095 EP-A 0 315 968 EP-A 0 318 081 EP-A 0 599 344 GB 8514349 Mendoza, J.A. Biotechnol. You > _n 10 (1991) 535 540 US Patent 4,808,705 WO 91/15509 WO 93/22335 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (12)

RE I V I ND I C AC I ONE S

1. Process perfected to prevent the formation of protein aggregates in a reconstituted lyophilisate of a protein pharmaceutical composition, according to which a buffered aqueous solution of the protein is frozen, thawed, divided into compartments of injectable amounts and said compartments they are lyophilized, and in which the buffered aqueous solution of the protein contains potassium phosphate buffer as the buffer substance and the ratio of potassium ions to sodium ions in the solution is 10: 1 or greater.

2. Method as claimed in claim 1, characterized in that the concentration of buffer in the compartment is between 10 mmol / liter and 300 mmol / l.

3. Method as claimed in claims 1-2, characterized in that the osmolarity of the reconstituted solution of the compartment is preferably between 100 and 500 mOsm, preferably 300 + 50 mOsm.

4. Process as claimed in claims 1-3, characterized in that the buffered aqueous solution is buffered in a pH range between 6-8. Process as claimed in claims 1-4, characterized in that the buffered aqueous solution contains a non-ionic detergent. 6. Process as claimed in claims 1-5, characterized in that the buffered aqueous solution contains a sugar at a concentration of 10-100 mg / ml. The method as claimed in claims 1-6, characterized in that the protein is an antibody. 8. Solid molten storage form of a protein with a low content of aggregates, characterized in that it is essentially amorphous, contains a frozen solution of the protein with potassium phosphate buffer as a buffer and the ratio of potassium ions to sodium ions It is at least 10: 1. 9. A solid storage form of a protein as claimed in claim 8, characterized in that the storage form is prepared by lyophilization. 10. Form of storage of a protein as claimed in claims 8 or 9, characterized in that the protein is an antibody. 11. Pharmaceutical composition of a protein in an aqueous solution buffered in the pH range between 6 and 8, characterized in that the solution contains potassium phosphate buffer as buffer substance, and a) the ratio of potassium ions to sodium ions in the solution is 10: 1 or greater, b) the buffer concentration is between 10 and 300 mmol / liter. 12. Pharmaceutical composition as claimed in claim 11, characterized in that the protein is an antibody.