Title:  Process for stabilizing proteins

United States Patent:  6,238,664

Assignee:  Boehringer Mannheim GmbH (Mannheim, DE)

Filed:  November 19, 1998

Foreign Application Priority Data:  Nov 22, 1997[EP] (97120528); Feb 19, 1998[EP] (98102846)

An aqueous protein solution buffered with a potassium phosphate buffer, in which the ratio of potassium ions to sodium ions in the solution is at least 10:1, is resistant to the formation of protein aggregates and particles under conditions of freezing, thawing, lyophilization, and reconstitution.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition comprising an aqueous buffered solution having a protein dissolved therein, wherein the solution contains potassium ions and either contains no sodium ions or contains sodium ions such that the ratio of potassium ions to sodium ions in the solution is at least 10:1. In an embodiment of this invention the solution is buffered with a potassium phosphate buffer in a concentration of from 10 to 300 mmol/liter in the solution. In a more specific embodiment, the concentration of the potassium phosphate buffer in the solution is from 50 to 250 mmol/liter. In an embodiment the solution is at a pH of from 6 to 8 when measured at a temperature of from 4^oC. to 30^oC. In a more specific embodiment the pH of the solution is from 6.5 to 7.5 when measured at a temperature of from 4^oC. to 30^oC. In an embodiment of this invention the ratio of potassium ions to sodium ions is at least 50:1. Preferably the buffer is substantially free of sodium ions.

This invention also provides a process for forming an aqueous buffered solution having a protein dissolved therein comprising: a) dissolving the protein in an aqueous solution; and b) adjusting the aqueous solution with a potassium phosphate buffer so that the aqueous solution having the protein dissolved therein contains potassium ions and either contains no sodium ions or contains sodium ions such that the ratio of potassium ions to sodium ions in the solution is at least 10:1. After the buffered solution is formed, it can be frozen or lyophilized if desired, in accordance with conventional techniques. In a further embodiments, the frozen solution is thawed and the lyophilized solution is reconstituted.

In an embodiment of the composition and process of this invention the solution is buffered with a potassium phosphate buffer in a concentration of from 10 to 300 mmol/liter in the solution. In a more specific embodiment, the concentration of the potassium phosphate buffer in the solution is from 50 to 250 mmol/liter. In an embodiment the solution is at a pH of from 6 to 8 when measured at a temperature of from 4^oC. to 30^oC. In a more specific embodiment the pH of the solution is from 6.5 to 7.5 when measured at a temperature of from 4^oC. to 30^oC. In an embodiment of this invention the ratio of potassium ions to sodium ions is at least 50:1. Preferably the buffer is substantially free of sodium ions.

In accordance with this invention, any protein can be utilized. The specific identity of the protein is not critical, provided that an aqueous solution of the protein can be formed. In an embodiment of this invention the protein is an antibody. Furthermore, the concentration of the protein in the solution is not critical. Any amount of protein which can be dissolved in the aqueous buffered solution can be utilized. Typically, the concentration of protein which is dissolved in solution will be from 1 mg/ml to 50 mg/ml.

This invention provides a process for preventing the formation of protein aggregates in a solution of a pharmaceutical composition of a protein, preferably of an antibody, that is reconstituted from a lyophilisate wherein an aqueous buffered solution of the protein is frozen, thawed, divided into compartments of injectable amounts and these compartments are lyophilized which is characterized in that the aqueous buffered solution of the protein contains potassium phosphate buffer as a buffer substance and the ratio of potassium to sodium ions in the solution is 10:1 or larger. The aqueous buffer solution preferably contains essentially no sodium ions.

The invention enables pharmaceutical compositions of proteins, in particular proteins which have a tendency to dimerize or multimerize such as antibodies, to be formulated into. a stable pharmaceutical composition in a neutral pH range (pH 6-8, preferably pH 6.5-7.5). Proteins such as antibodies tend to aggregate in the neutral pH range especially if the solutions are frozen (optionally lyophilized) once or several times and thawed again.

A pharmaceutical composition is especially advantageous in potassium phosphate buffer in the pH range between 6 and 8, at a buffer concentration between 10 and 300 mmol/l, preferably between 50 and 250 mmol/l in which the lowest possible number of sodium ions are present in the pharmaceutical composition. A suitable ratio of potassium to sodium ions in the solution is 10:1 or more. It is particularly preferable that potassium phosphate buffer is used alone as the buffer substance in the pharmaceutical composition and no sodium salt (such as e.g. sodium chloride) is added. In such a case almost no sodium ions are present in the pharmaceutical composition or it only contains them in such low amounts that they do not cause formation of aggregates of proteins during repeated freezing or thawing.

It has turned out that lyophilisates of protein solutions which have been frozen at least once during the production process can then be reconstituted substantially without formation of turbidities if potassium phosphate buffer is used as the buffer substance. The usual buffers such as sodium phosphate buffer, histidine buffer or citrate buffer lead to the formation of aggregates in such a 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 through below ca. -15^oC., have eutectic points above ca. -15^oC. and can thus already be stored at this temperature or at lower temperatures preferably e.g. at -20^oC. Since a solution is only completely frozen through below the eutectic temperature, this means that a protein in a phosphate buffer containing sodium ions is subjected to a higher stress during the frozen storage (usually at -20^oC.) and during the freezing/thawing process than in a buffer free of sodium ions or in a buffer in which the sodium ion concentration is very low. According to the invention this stress is avoided in the above-mentioned formulations resulting in a suppression of aggregate and particle formation. This formulation enables a stable storage of the protein solution at -20^oC. which can save costs. Potassium phosphate buffers in contrast to sodium phosphate buffers have only a slight pH shift (preferably at most .+-.1 pH unit, particularly preferably at most .+-.0.5 pH units) during the freezing process.

It has turned out that the concentration of the phosphate buffer should be at least 10 mmol/l, preferably about 50 mmol/l or higher in order to effectively prevent particle formation. Since the osmolarity should not be too high (it should advantageously be in the physiological range, preferably ca. 300 mOsm after reconstitution (.+-.20 mOsm, a range of 100 to 500 mOsm is also suitable)) in pharmaceutical compositions (i.e. preferably in the reconstituted solution), the concentration of the buffer substance or optionally the sum of buffer substance and salt should be not more than 250-300 mmol/l. The buffer concentration is preferably between 50 and 250 mmol/l in the compartment. However, higher concentrations of buffer substance and/or salt can be tolerated in the production of the solutions (bulkware) used to produce the compartments.

If a salt additive is desired in the pharmaceutical composition especially to adjust the ionic strength, it is advantageous according to the invention to also not use sodium salts or to select a concentration of the sodium ions which is substantially lower than the concentration of the potassium ions. It is therefore expedient to add a potassium salt such as potassium chloride instead of the otherwise usual sodium chloride. However, it has turned out that low amounts of sodium salts (e.g. ca. 10 mmol/l or less) do not interfere provided the ratio of potassium ions to sodium ions is 10:1 or higher. It is not possible to add calcium salts such as e.g. calcium chloride since calcium phosphate is precipitated by such an addition and hence, apart from the formation of undesired turbidity, the buffer effect of the potassium phosphate according to the invention is abolished.

Non-soluble aggregates whose formation should be prevented in the process according to the invention are essentially understood as protein aggregates whose size is usually at least 1 .mu.m but can also be in the range above 10 .mu.m. The particles can be determined by suitable particle counting methods using commercial particle counting instruments such as e.g. the particle counting instrument AccuSizer 700 from PSS (Particle Sizing Systems, USA). According to the invention an improvement of the process is achieved when the number of particles between 2 and 400 .mu.m/ml is <3000 or the number of particles between 10 and 400 .mu.m/ml is 2000 or less. According to the USP (US-Pharmacopoeia) a maximum of 6000 particles in the range above 10 .mu.m and a maximum of 600 particles in the range above 25 .mu.m are allowed per injected dose of a pharmaceutical preparation. This can be achieved according to the invention in a simple manner for therapeutic compositions of proteins.

In accordance with this invention any protein can be utilized. The invention is based on the use of the aqueous buffered solution in accordance with this invention, and is not limited as to the specific protein dissolved therein. Proteins (polypeptides) are understood within the sense of the invention as naturally occurring and recombinant proteins or protein fragments as well as chemically modified proteins and proteins containing amino acid substitutions and additions. Proteins which are desirably 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.

Compartments within the sense of the invention are understood as aliquots of the protein solution which, optionally after further processing (addition of further pharmaceutically acceptable substances), are suitable as pharmaceutical compositions preferably for injection in 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 range (ca. pH 6-8, preferably about pH 7).

According to the invention it is preferable to add a nonionic detergent such as polysorbate (e.g. Tween.RTM. 80), 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 cryoprotectors or glass formers such as a non-reducing sugar (preferably sucrose or trehalose), advantageously at a concentration of at least 10 mg/ml, preferably of ca. 30-100 mg/ml.

Consequently a further subject matter of the invention is a low aggregate, meltable solid storage form of a protein which is essentially amorphous and is composed of a frozen solution of the protein and potassium phosphate buffer as the main buffer substance in which the ratio of potassium ions to sodium ions in the solution is at least 10:1.

Independent of the concentration of potassium ions and the residual content of sodium ions, the ratio of potassium to sodium ions should be at least 10:1, preferably at least 50:1. It is particularly preferable to use essentially sodium-ion-free potassium buffer.

In a further preferred embodiment of the invention the pharmaceutical composition contains a protein which has- been produced by an in vitro cell culture (for example recombinant production or culture of a hybridoma cell line to produce monoclonal antibodies). In this case it is expedient to either add potassium salt and/or potassium phosphate buffer with the first addition of salt or/and buffer, or to rebuffer at a later time in the isolation and purification process. This enables the interim stable storage of the polypeptide preparation below 0^oC. Rebuffering is understood as an exchange of ions for example by dialysis. In the purification and isolation process of the protein the buffer or salt concentration can indeed be higher than 50-100 mmol/l before compartmentation since these compositions are not used therapeutically. However, it is essential that an osmolarity that is suitable for an injectable composition is adjusted before the compartmentation.

The disclosure of European Patent Application No. 97120528.1 is incorporated herein by reference.

Claim 1 of 45 Claims

What is claimed is:

1. In a process for reconstituting a lyophilizate formed from an aqueous solution having a protein dissolved therein, the improvement comprising:

wherein the solution contains potassium ions and either contains no sodium ions or contains sodium ions such that the ratio of potassium ions to sodium ions in the solution is at least 10:1; and

the solution being buffered at a pH from 6 to 8 with a potassium phosphate buffer in a concentration of from 10 to 300 mmol/liter in the solution.

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