Pharmaceutical formulation and preparation method thereof

Abstract

To provide an etanercept pharmaceutical composition, a method for producing the same, and a method for treating a patient having rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or psoriasis.SOLUTION: A pharmaceutical composition comprising 75mM to 150mM NaCl, 5mM to 100mM arginine, 0.5% to 2% (w / v) sucrose, and 40mg to 100mg / mL etanercept, wherein the composition comprises 2. less than 0mM total additional buffer, and wherein the pH of the composition is between 6.1 and 6.5.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 411,458, filed October 21, 2016, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a formulation of an etanercept pharmaceutical composition. The present invention also relates to a method for removing a buffer and a method for formulating an etanercept pharmaceutical composition. [Background technology]

[0003] Formulation of protein drugs can present many challenges to pharmaceutical scientists. A formulation must be found that stabilizes the protein drug and resists degradation due to protein degradation, aggregation, misfolding, etc. Finding appropriate stability conditions can be a challenge, particularly for engineered proteins that differ substantially from known proteins. It is also desirable to provide protein drugs in a format that is convenient for patients. Desired properties include stability at ambient and refrigerated temperatures, suitability for long-term storage, appropriate dosing frequency and amount, and minimal discomfort upon administration.

[0004] Etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75-kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, CH3 domain, and hinge region of IgG1, but not the CH1 domain. When expressed in mammalian cells, it forms a homodimeric complex with two domains of the TNF receptor. It is therefore an artificial protein distinct from both antibodies and soluble TNF receptors and, therefore, undergoes a different degradation pathway than either. Etanercept is commercially available as ENBREL® (Amgen Inc, Thousand Oaks, CA) and is approved for the treatment of moderate to severe active rheumatoid arthritis, moderate to severe active polyarticular juvenile idiopathic arthritis (JIA) in patients 2 years of age and older, chronic moderate to severe plaque psoriasis (PsO) in adults, psoriatic arthritis (PsA) in adults, and active ankylosing spondylitis (AS). Etanercept was initially available as a lyophilized formulation that required reconstitution immediately prior to injection.

[0005] When the lyophilized product is reconstituted with water for injection, the formulation is approximately 25 mg / mL, 10 mM Tris-HCl, 4% mannitol, 1% sucrose, and pH 7.4. However, this formulation is not stable during storage. It has been found that a liquid formulation of etanercept can be achieved using arginine to stabilize the protein (see U.S. Patent No. 7,648,702). An exemplary liquid formulation consists of an aqueous solution of 50 mg / mL etanercept, 25 mM phosphate buffer, 25 mM L-arginine hydrochloride, 100 mM NaCl, and 1% sucrose, pH 6.3. Summary of the Invention [Means for solving the problem]

[0006] This specification provides a new and improved formulation of etanercept. In particular, the present invention provides a pharmaceutical composition containing etanercept that is stable and can be conveniently stored as a liquid for a long period of time at controlled room temperature (CRT) without the presence of additional buffering agents. Furthermore, when the pharmaceutical compositions of the present invention are injected into a subject, they also show a significant reduction in injection pain compared with commercially available conventional formulations. Therefore, these pharmaceutical compositions are more convenient and advantageous for patients.

[0007] Another aspect of the present invention provides a method for formulating a pharmaceutical preparation of etanercept at a desired pH, but without the presence of additional buffering agents in the final formulation.

[0008] In another aspect, the present invention provides a pharmaceutical composition comprising etanercept, NaCl, arginine, and sucrose, substantially free of additional buffering agents and having a pH of 6.1 to 6.5. In one embodiment, the pharmaceutical composition can maintain a pH of 6.1 to 6.5 after storage at controlled room temperature (CRT) for two weeks. In another embodiment, the etanercept concentration is 40 mg / mL to 100 mg / mL. In another embodiment, the pharmaceutical composition is isotonic. In another embodiment, the pharmaceutical composition comprises 20 mM to 150 mM NaCl, 5 mM to 100 mM arginine, and 0.5% to 2% (w / v) sucrose. In another embodiment, the pharmaceutical composition comprises a surfactant. In another embodiment, the surfactant is polysorbate 20, polysorbate 80, or poloxamer 188. In another embodiment, the surfactant is polysorbate 20 at a concentration of 0.001% to 0.1% (w / v). In another embodiment, the surfactant is polysorbate 80 at a concentration of 0.001% to 0.1% (w / v). In another embodiment, the surfactant is polysorbate 188 at a concentration of 0.01% to 0.3% (w / v). In another embodiment, the pharmaceutical composition maintains a pH of 5.8 to 6.7 for at least two weeks when stored at about 25°C, and less than 6% of the total etanercept is aggregated in a high molecular weight form as assessed by size exclusion chromatography. In another embodiment, the pharmaceutical composition maintains a pH of about 6.1 to about 6.5. In another embodiment, less than 28% of the total etanercept is in a misfolded form as assessed by hydrophobic interaction chromatography. In another embodiment, the pharmaceutical composition consists essentially of about 40 to 100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water. In another embodiment, the pharmaceutical composition consists essentially of about 40-100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, about 0.01% polysorbate 20, and water.

[0009] In another aspect, the present invention provides a method for formulating an etanercept pharmaceutical composition to remove additional buffering agents and maintain a pH of 6.1 to 6.5, the method comprising the steps of: formulating an etanercept formulation into a formulation containing additional buffering agents at a pH of 6.1 to 6.5; exchanging the formulation containing additional buffering agents with a formulation containing no additional buffering agents at a pH of 5.6 to 6.5; and recovering the resulting pharmaceutical formulation. In one embodiment, the exchanging step uses diafiltration. In another embodiment, the formulation without additional buffering agents is isotonic. In another embodiment, the formulation without additional buffering agents contains sucrose, arginine, and NaCl. In another embodiment, the formulation without additional buffering agents contains 20 mM to 150 mM NaCl, 5 mM to 100 mM arginine, and 0.5% to 2% (w / v) sucrose. In another embodiment, the formulation without additional buffering agents consists essentially of about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water. In another embodiment, the method of formulating the etanercept pharmaceutical composition further comprises adding a polysorbate. In another embodiment, the polysorbate is polysorbate 20 at a concentration (w / v) of 0.001% to 0.1%. In another embodiment, the method of formulating the etanercept pharmaceutical composition further comprises filtering the pharmaceutical composition. In another embodiment, the method of formulating the etanercept pharmaceutical composition further comprises aliquoting the pharmaceutical composition into drug product forms.

[0010] In another aspect, the present invention provides a kit comprising the above-described etanercept pharmaceutical composition in pharmaceutical product form and instructions for storage and use.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising etanercept, NaCl, arginine, sucrose, phosphate buffer, and benzyl alcohol, the composition having a pH of 6.1 to 6.5. In one embodiment, the benzyl alcohol is present at a concentration (v / v) of 0.1% to 5.0%. In another embodiment, the benzyl alcohol is present at a concentration of about 0.9%. In another embodiment, the pharmaceutical composition comprising etanercept further comprises polysorbate 20 at a concentration (w / v) of 0.001% to 0.1%. In another embodiment, the polysorbate 20 is present at a concentration of about 0.004%. In another embodiment, the pharmaceutical composition comprising etanercept consists essentially of about 40 to 100 mg / mL etanercept, about 25 mM arginine, about 100 mM sodium chloride, about 1% (w / v) sucrose, about 25 mM phosphate buffer, and about 0.9% (v / v) benzyl alcohol. In another embodiment, the pharmaceutical composition comprising etanercept consists essentially of about 40 to 100 mg / mL etanercept, about 25 mM arginine, about 100 mM sodium chloride, about 1% (w / v) sucrose, about 25 mM phosphate buffer, about 0.9% (v / v) benzyl alcohol, and about 0.004% (w / v) polysorbate 20.

[0012] In another aspect, the present invention provides a single-dose container containing the above-described pharmaceutical composition comprising etanercept. In one embodiment, the pharmaceutical composition consists essentially of about 40-100 mg / mL etanercept, about 25 mM arginine, about 100 mM sodium chloride, about 1% (w / v) sucrose, about 25 mM phosphate buffer, and about 0.9% (v / v) benzyl alcohol. In another embodiment, the pharmaceutical composition consists essentially of about 40-100 mg / mL etanercept, about 25 mM arginine, about 100 mM sodium chloride, about 1% (w / v) sucrose, about 25 mM phosphate buffer, about 0.9% (v / v) benzyl alcohol, and about 0.004% (w / v) polysorbate 20. In another embodiment, the single-dose container is a vial, syringe, or autoinjector. In another embodiment, the single-dose container contains an aqueous formulation consisting of 50.0 mg / mL etanercept, 120 mM sodium chloride, 25 mM L-arginine, 1.0% (w / v) sucrose.

[0013] In another aspect, the present invention provides a method for preparing a single-dose container containing a pharmaceutical composition comprising etanercept as described above, the method comprising filling, under sterile conditions, approximately a single dose of the pharmaceutical composition into the single-dose container. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the percent HMW (peak B) detected by SEC in the stability analysis of etanercept in Example 3. [Figure 2] FIG. 2 shows the percent LMW detected by dSEC in the stability analysis of etanercept in Example 3. [Figure 3] FIG. 3 shows the percent peak 3 detected by HIC in the stability analysis of etanercept in Example 3. [Figure 4] FIG. 4 shows the percent peak 3 detected by HIC in the stability analysis of etanercept stored in stainless steel cryocontainers in Example 4. [Figure 5]FIG. 5 shows the percent LMW detected by dSEC in the stability analysis of etanercept stored in stainless steel cryocontainers in Example 4. [Figure 6] FIG. 6 shows the percent Peak B detected by SEC in the stability analysis of etanercept stored in stainless steel cryocontainers in Example 4. [Figure 7] FIG. 7 shows the percent Peak B detected by SEC in the stability analysis of freeze-thawed etanercept in Example 4. [Figure 8] FIG. 8 shows the pH stability at controlled room temperature (CRT) of the AEX intermediate pool prepared in the analysis of Example 6. [Figure 9] FIG. 9 shows the stability of pH (A) and conductivity (B) of UF / DF pools at CRT for the analysis of Example 6. [Figure 10] FIG. 10 shows the pH (A) and conductivity (B) stability of etanercept formulated in SAS solution in the analysis of Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides improved pharmaceutical compositions of etanercept. As used herein, the term "pharmaceutical composition" is understood to refer to a polypeptide formulation suitable for injection and / or administration to a patient in need thereof. More specifically, the pharmaceutical composition is substantially sterile and does not contain any agents that are excessively toxic or infectious to the recipient. Etanercept is a soluble form of the p75 TNF receptor fused to the Fc domain of human IgG1 (TNFR:Fc). Commercially available etanercept is known as ENBREL® (Immunex Inc., Thousand Oaks, CA). Etanercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons (Physicians' Desk Reference, 2002, Medical Economics Company Inc.). The complete sequence expressed in CHO cells is shown below: However, it should be understood that minor modifications and deletions (up to 10%) may be possible to make to this sequence and may be used within the scope of the present invention. [ka] [ka]

[0016] The present invention provides pharmaceutical compositions containing etanercept but substantially free of additional buffering agents. The phrase "additional buffering agent" refers to a component of an etanercept composition or formulation, other than etanercept itself, that significantly contributes to the buffering capacity of the composition or formulation. Etanercept itself has been shown herein to provide all the buffering necessary to maintain a pH between 6.1 and 6.5, particularly about 6.2 to 6.3, under the conditions described below. As shown in Example 1 below, this pH range was found to be effective in maintaining the desired stability characteristics of etanercept formulations (less than 6% high molecular weight aggregates and less than 28% misfolded and clipped species).

[0017] The phrase "substantially free of additional buffering agents" means that buffering agents other than etanercept are present at less than 0.5 mM. The phrase "total additional buffering agents" collectively refers to all components of an etanercept composition or formulation, other than etanercept itself, that significantly contribute to the buffering capacity of the composition or formulation. In some embodiments, a pharmaceutical composition according to the present invention contains less than 2.0 mM total additional buffering agents, less than 1.5 mM total additional buffering agents, less than 1.0 mM total additional buffering agents, less than 0.5 mM total additional buffering agents, less than 0.25 mM total additional buffering agents, less than 0.1 mM total additional buffering agents, or less than 0.05 mM total additional buffering agents. In typical pharmaceutical compositions, additional buffering agents, often at concentrations of 5.0 mM or greater, are used to maintain the pH in the desired range. Various well-known additional buffering agents include histidine, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (Tris), various forms of acetate, and diethanolamine. One common buffering agent is sodium phosphate because of its buffering capacity at or near pH 6.2. Sodium phosphate is the buffering agent used in currently commercially available liquid formulations of etanercept (because the desired pH of the liquid formulation is 6.3). The invention described herein is substantially free of sodium phosphate in the etanercept pharmaceutical formulation. Surprisingly, despite being substantially free of any additional buffering agents, the pH of the pharmaceutical compositions of the present invention is maintained between 6.1 and 6.5, even after long-term storage. Even more surprisingly, when injected into a subject (e.g., a human subject or patient), pharmaceutical compositions substantially free of additional buffering agents provide significantly more pain relief than currently commercially available buffered formulations. Although phosphate is often chosen as a buffering agent in pharmaceutical compositions because it has a near-neutral pH buffering capacity and is considered one of the less painful buffering components (e.g., compared to citrate buffers), the inventors have determined that phosphate buffers around pH 6.3 cause pain upon injection.

[0018] Unless otherwise clear from the context in which it is used, a "formulation solution" or "formulation buffer" is a solution or buffer that does not itself contain etanercept and is used to make a formulation that contains etanercept.

[0019] Typically, the concentration of etanercept in the pharmaceutical composition of the present invention is about 40 mg / mL to about 200 mg / mL in an aqueous formulation (e.g., water as a solvent). More preferably, the concentration of etanercept is about 40 mg / mL to about 100 mg / mL, even more preferably about 40 mg / mL to about 75 mg / mL, and optionally about 50 mg / mL.

[0020] The pharmaceutical compositions of the present invention also contain arginine. Arginine has been shown to substantially contribute to the stabilization of etanercept in liquid formulations (see U.S. Pat. No. 7,648,702, incorporated herein by reference). Pharmaceutically suitable forms of arginine are commercially available. L-arginine (e.g., L-arginine HCl or L-arginine base) is generally the arginine used in pharmaceutical formulations. It is recognized that within the pH range of 6.0 to 6.6, particularly at a pH of about 6.2 to 6.3, arginine does not significantly contribute to the buffering capacity of the formulation. Therefore, it is not an additional buffering agent in the etanercept formulations or compositions of the present invention. The concentration of arginine in the compositions of the present invention is preferably about 1 mM to about 1 M, more preferably about 10 mM to about 200 mM, or about 5 mM to about 100 mM, more preferably about 10 mM to about 100 mM, even more preferably about 15 mM to about 75 mM, and even more preferably about 25 mM. Thus, in one embodiment of the present invention, a pharmaceutical composition comprises about 50 mg / mL to 75 mg / mL of etanercept and about 25 mM of arginine, the pharmaceutical composition being substantially free of additional buffering agents, and the pH of the composition is 6.0 to 6.6. As used herein, the term "about" is understood to mean that the component concentrations of the described formulations may vary by up to and including 10% of a given value. For example, if a formulation has about 10 mg / mL of polypeptide, this is understood to mean that the formulation may have 9 to 11 mg / mL of the described polypeptide.

[0021] The pharmaceutical composition may contain additional excipients, as long as the excipient is not an additional buffer, particularly a phosphate buffer. Examples of additional excipients of the present invention include, but are not limited to, sugars / polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, etc.; polymers such as serum albumin (bovine serum albumin (BSA), human SA, or recombinant HA), dextran, PVA, hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); polyhydric alcohols (e.g., PEG, ethylene glycol, and glycerol), non-aqueous solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF); amino acids such as proline, L-serine, alanine, glycine, lysine hydrochloride, sarcosine, and gamma-aminobutyric acid, and surfactants.

[0022] In some preferred embodiments of the present invention, the excipient comprises NaCl and / or sucrose. NaCl may be present in the pharmaceutical composition at a concentration of about 5 mM to about 200 mM, more preferably about 20 mM to about 150 mM, and even more preferably about 80 mM to about 140 mM. Sucrose may be added to a concentration of about 0.5% to about 2% (w / v) sucrose, more preferably about 0.8% to about 1.2% (w / v) sucrose, and even more preferably about 1% (w / v) sucrose.

[0023] The osmolality of a pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and minimize discomfort to the patient upon administration. It is generally preferred that the pharmaceutical composition be isotonic with serum, i.e., have the same or similar osmolality, which is achieved by the addition of an osmolality-adjusting agent. Serum has an osmolality of about 300 + / - 50 millimolar osmolality per kilogram; therefore, the osmolality of an isotonic pharmaceutical composition would be about 180 to about 420 millimolar osmolality. In some embodiments, this range would be about 250 to about 350 millimolar osmolality.

[0024] An osmolality adjusting agent is understood to be a molecule that contributes to the osmolality of a solution. Examples of osmolality adjusting agents suitable for adjusting osmolality include, but are not limited to, amino acids (e.g., arginine, cysteine, histidine, and glycine), salts (e.g., sodium chloride, potassium chloride, and sodium citrate), and / or sugars (e.g., sucrose, glucose, and mannitol). The concentration of the osmolality adjusting agent in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM. In some embodiments, the concentrations of NaCl and sucrose are adjusted to produce an isotonic pharmaceutical composition. In some embodiments, as exemplified below, the pharmaceutical composition contains about 40 to 100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water. In particular, the pharmaceutical composition may consist essentially of about 50 to 100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, about 0.01% polysorbate 20, and water.

[0025] Optionally, the pharmaceutical composition of the present invention may contain a surfactant. A surfactant is an agent that reduces solution / surface-induced stress. Examples of surfactants include polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 (e.g., TWEEN-20® (Sigma-Aldrich, St. Louis, MO) or TWEEN-80® (Sigma-Aldrich, St. Louis, MO)), sodium dodecyl sulfate (SDS), polyoxyethylene copolymers, poloxamers such as poloxomer 188 (e.g., PLURONIC® F-68 (Sigma-Aldrich, St. Louis, MO)) or poloxomer 407 (e.g., PLURONIC® F-127 (Sigma-Aldrich, St. Louis, MO)), CHAPS, monolaurate, or any combination of the above. A preferred surfactant is polysorbate 20. For example, polysorbate 20 can be included in the pharmaceutical composition at a concentration of about 0.001% to about 0.03% (w / v). In certain embodiments shown below by way of example, polysorbate 20 can be included in the pharmaceutical formulation at a concentration of 0.01% or about 0.004% (w / v).

[0026] Pharmaceutical Composition Testing The following examples illustrate how one skilled in the art can determine whether a formulation can maintain a pH in a desired range. Essentially, a pharmaceutical composition is formulated and stored in a test container (which may be a glass vial, glass syringe, plastic syringe, stainless steel container, or any type of sterile device suitable for pharmaceutical compositions), and the pH is evaluated at time 0 and, if necessary, at the indicated times thereafter. Typically, the test conditions will predict and focus on the storage needs of the pharmaceutical composition. For example, the formulations of the present invention can maintain a desired pH for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, and at least 24 weeks under controlled room temperature (CRT). CRT is defined by the USP to encompass the normal customary working environment of 20°C to 25°C (68°F to 77°F); resulting in a mean dynamic temperature calculated to be 25°C or less; and having a thermostatically maintained temperature that allows for excursions between 15°C to 30°C (59°F to 86°F) experienced in pharmacies, hospitals, and warehouses.

[0027] In one embodiment, the pharmaceutical compositions of the present invention exhibit certain quality characteristics. Tests for these quality characteristics are also described below in the Examples. For example, the pharmaceutical compositions of the present invention contain less than 6% of total etanercept aggregated into high molecular weight forms when assessed using size exclusion chromatography. In another example, the pharmaceutical compositions of the present invention contain less than 28% of total etanercept in misfolded forms when assessed using hydrophobic interaction chromatography.

[0028] The pharmaceutical compositions of the present invention may retain stability by maintaining pH and / or other quality characteristics of interest (minimal high molecular weight forms and minimal misfolded forms) at the following temperatures and for extended periods: (1) at -30°C (frozen) for at least 4 weeks, at least 3 months, at least 6 months, at least 12 months, and at least 36 months; (2) at one freeze / thaw cycle, up to two freeze / thaw cycles, up to three freeze / thaw cycles, and up to five freeze / thaw cycles; (3) at 4°C (refrigerated temperature) for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, and at least 52 weeks; (4) at 25°C (room temperature) for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks; and (5) at 40°C (accelerated stability testing) for at least 2 weeks.

[0029] The pharmaceutical compositions of the present invention also surprisingly reduce pain upon injection in subjects. This property can be assessed using a visual analog scale (VAS), as validated by Gallagher et al., 2002, Am. J. Em. Med. v20;i4:287-290. A trained medical professional administered the drug via injection, and within 30 seconds of each injection, subjects rated the level of injection pain using a 100mm visual analog scale (VAS). A difference of 13-16mm on the VAS is considered clinically meaningful. Using this approach, a phosphate-free placebo formulation demonstrated significantly greater pain reduction than a phosphate-containing placebo formulation at pH 6.3 and a current marketed formulation containing both etanercept and phosphate at pH 6.3.

[0030] The production and purification of etanercept for use in the pharmaceutical compositions and methods of the present invention can be carried out by any standard method. Generally, etanercept is recombinantly expressed in CHO cells and secreted into the culture medium. The culture medium is collected, filtered, and purified, for example, using various chromatographic techniques. For example, protein A can be used to purify Fc domain-containing polypeptides, such as etanercept, and is advantageous as an initial processing step. Other techniques for purifying polypeptides include fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSET™ chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, as well as any combination of known or later-discovered purification techniques. Examples of useful preparation and purification techniques can be found in U.S. Pat. Nos. 7,294,481 (Fung), 7,452,695 (Van Ness et al.), 7,122,641 (Vedantham et al.), 7,157,557 (Sassenfeld et al.), 7,300,773 (Drapeau et al.), 8,163,522 (Brockhaus et al.), and 7,648,702 (Gombotz et al.).

[0031] Methods of the Invention The present invention also provides a method for formulating an etanercept pharmaceutical composition to remove a buffer and maintain a pH within a target range, the method comprising formulating etanercept in a buffered formulation at a target range, exchanging the buffered formulation for a non-buffered formulation within or just below the target range, and recovering the resulting pharmaceutical formulation of etanercept. In a preferred embodiment, as exemplified below, the method comprises formulating the etanercept formulation in a buffered formulation at a pH of 6.0 to 6.6, exchanging the buffered formulation for a non-buffered formulation at a pH of 5.6 to 6.5, and recovering the resulting pharmaceutical formulation. To obtain a non-buffered etanercept composition that maintains a pH of 6.1 to 6.5, it is important to ensure that the pH of both the starting buffered etanercept formulation and the non-buffered formulation are adjusted. For example, if the starting buffered etanercept formulation has a pH of 7.2, it would be adjusted to a pH range of 6.1 to 6.5 with a strong acid such as HCl. Similarly, an unbuffered formulation used for replacement should be titrated to a pH of 5.6 to 6.5. Care should be taken during titration because the unbuffered formulation used for replacement does not have a buffer.

[0032] To exchange a buffered formulation for an unbuffered formulation, those skilled in the art can use various buffer exchange techniques well known in the art. Dialysis utilizes selective diffusion through a semipermeable membrane to remove relatively small, unwanted molecules from a relatively large protein formulation. In one embodiment, successive equilibrations are performed until the concentration of the unwanted molecules is reduced by a desired factor. For example, three successive equilibrations can be performed, each with a dilution of 100-fold or more, to achieve a concentration reduction of 1,000,000-fold or more. Ultrafiltration and diafiltration are similar to dialysis in that they use semipermeable membranes. However, unlike the passive diffusion of dialysis, ultrafiltration and diafiltration involve forcing a solution through a membrane using various techniques. Pressure and centrifugation are commonly used. Yet another method of buffer exchange can be performed using gel filtration or size exclusion chromatography. Many other chromatographic techniques, such as ion exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are also well within the skill of those skilled in the art and can be used to achieve buffer exchange.

[0033] After the buffered formulation has been exchanged for a non-buffered formulation, the method of the present invention includes recovering the resulting pharmaceutical formulation. At this point, substantially all of the buffer has been removed, but the pH is still maintained at the desired level. For pharmaceutical compositions containing etanercept, the pH is maintained at 6.0 to 6.6.

[0034] The pharmaceutical formulation may be further processed, if necessary. For example, a surfactant may be added. In another example, the pharmaceutical composition may be filtered if particulate removal is desired. Alternatively, or in addition, the method of the present invention may also include aliquoting the pharmaceutical composition into drug product forms. Such drug product forms are distributed for end use by patients or healthcare providers. The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular, intravenous, intraperitoneal, intracerebrospinal, intraarticular, intrasynovial, and / or intrathecal administration. Parenteral administration may be by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be provided in unit dose form, e.g., ampoules or multi-dose containers. Pharmaceutical compositions may be provided in vials, packs, or dispenser devices, which may contain one or more unit dose forms containing the active ingredient, if desired. In one embodiment, the dispenser device may include a syringe with a single dose of the liquid formulation, ready for injection. In another embodiment, the pharmaceutical composition is aliquoted into cassette components for use with a reusable autoinjector. In yet another aspect of the invention, the pharmaceutical composition may be provided packaged within or together with a wearable injection device, hi yet another embodiment, the pharmaceutical composition may be aliquoted into a drug product form suitable for a needle-free injection device.

[0035] The pharmaceutical composition can also be divided into suitable formats as depot preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the preparation can be modified with suitable polymers or hydrophobic materials (e.g., as an emulsion in an acceptable oil), or ion exchange resins, or can be converted into sparingly soluble derivatives, for example, sparingly soluble salts.

[0036] In another embodiment, the present invention relates to a kit or container containing the pharmaceutical composition of the present invention. The kit can also include instructions for storing and using the pharmaceutical composition. The container can be, for example, a disposable container, i.e., a container containing a single-dose formulation of the present invention. The disposable container can contain a single dose plus enough excess to ensure that a specified single dose can be administered to a patient from the container, but it is recognized that there is not enough excess so that the container can be used to administer a second dose. Examples of containers suitable for use in some aspects of the present invention (whether they are disposable or reusable) include vials, syringes, and autoinjectors. Examples of suitable autoinjectors include those found in U.S. Pat. Nos. 8,177,749, 8,052,645, and 8,920,374, U.S. Patent Application Nos. 12 / 993163, 13 / 269750, 13 / 454531, 14 / 112479, 14 / 777255, and 14 / 777259, and WO 2014 / 0089393, WO 2016 / 033496, and WO 2016 / 033507, each of which is incorporated by reference in its entirety.

[0037] The etanercept-containing compositions and formulations of the present invention, as well as the syringes, autoinjectors, kits, etc. described herein, can be used to treat patients with diseases that benefit from etanercept treatment. Examples of such diseases include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and psoriasis. Methods for treating patients with etanercept are described, for example, in U.S. Patent Nos. 7,915,225, 8,119,605, 8,410,060, 8,722,631, and 8,119,604 (each of which is incorporated herein by reference in its entirety).

[0038] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. [Example]

[0039] Example 1: Stability test of various formulations This example demonstrates the effect of pH and buffer on 50 mg / mL etanercept and evaluates the stability of a high concentration (100 mg / mL) solution without added phosphate buffer. The following formulations were tested:

[0040] [Table 1]

[0041] Materials: PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) containing 50 mg / mL Enbrel drug substance was used in this study. For acetate- and buffer-free formulations, the material was dialyzed into a new formulation (no polysorbate) and concentrated to 50 mg / mL using a 10,000 MWCO centriprep. A sample of 50_SAS_100NaCl was also concentrated to 100 mg / mL (100_SAS_100NaCl). Benzyl alcohol was added to the current formulation to a final concentration of 0.9%. A freshly prepared 1% stock solution of polysorbate 20 was added to all formulations to a final concentration of 0.004%. All formulations were manually filled to a volume of 0.5 mL into 1 mL long BD glass syringes and then stoppered using an ASPU vacuum stopper device.

[0042] Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler Inlab MicroProbe. Samples were warmed to room temperature before measurement. Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 μL of sample, and a 290 osmolality standard was tested to ensure the system was operating properly. Size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software. Denaturing size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software.

[0043] Results: The pH of all formulations was maintained over 24 weeks.

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] [Table 5]

[0048] [Table 6]

[0049] [Table 7]

[0050] [Table 8]

[0051] Conclusions: During long-term storage at 25°C and 40°C, the lower pH formulations, A45SuT, A52SuT, and A58SuT, exhibited undesirable levels of low molecular weight, degraded, or clipped species as analyzed by denaturing size-exclusion chromatography. The higher-concentration formulation, 100_SAST_100NaCl, stored at 25°C and 40°C began to exhibit an increase in high molecular weight aggregates as analyzed by size-exclusion chromatography, but at 4°C, behaved similarly to the current commercial formulation. PASST+BeOH (which is the current commercial formulation modified by the addition of 0.004% polysorbate 20 and 0.9% benzyl alcohol) behaved similarly to the current commercial formulation at both 4°C and 25°C, but showed an increase in high molecular weight species as analyzed by SE-HPLC at later time points when stored at the elevated temperature of 40°C. However, the 50_SAST_100NaCl formulation maintained concentrations of high and low molecular weight species comparable to the current commercial formulation at all temperatures, even in the absence of phosphate buffer.

[0052] Example 2: Pain Study The study was a single-center, randomized, single-blind, crossover design in which 48 healthy men and women received a single SC injection of six solutions.

[0053] Six unique groups of eight subjects randomly assigned to each group were administered the test formulation (detailed in Table 9 below) by trained medical professionals. Injections were administered into each quadrant of the anterior abdominal wall, approximately one hour apart. Within 30 seconds of each injection, subjects rated the intensity of injection pain using a 100mm visual analog scale (VAS). Adverse events were collected from the start of the first injection until 30 days after the first injection. Safety follow-up phone calls were conducted on Day 2 (24 hours after the sixth injection) and Day 31 (±2 days).

[0054] [Table 9]

[0055] Statistical Methods: All analyses were performed on the safety analysis set, which consisted of all subjects who received at least one solution. A sample size of 48 subjects (8 per group) was selected to provide 93.4% power to detect a 15 mm difference between the solutions (α = 0.05, two-sided). A difference of 13 to 16 mm on the VAS is considered clinically meaningful (Gallagher et al., 2002, Am. J. Em. Med. v20;i4:287-290).

[0056] Summary statistics (mean, SD, standard error [SE], median, minimum, maximum) were calculated for the VAS scores by solution. VAS scores were analyzed using an analysis of variance (ANOVA) model, which included group, solution, and period as independent variables and subjects within groups as random effects. No adjustment for multiple comparisons was made.

[0057] The mean differences in VAS scores for the primary and secondary comparisons, the corresponding 95% confidence intervals (95% CI), and p values ​​were obtained.

[0058] [Table 10]

[0059] Conclusions: Both Solution C (non-product-specific placebo containing benzyl alcohol) and Solution D (non-product-specific placebo without sodium phosphate) had significantly lower mean VAS scores than Solution B (etanercept placebo; p<0.001), indicating relatively low injection site pain for these two solutions. No significant differences in mean VAS scores were found between Solutions C and D, between Solution B (etanercept placebo) and Solution F (active etanercept), or between the different injection volumes (0.51 and 1.0 mL). Solution A (negative pain control) caused the least pain compared with all other solutions. Seven subjects experienced one or more adverse events. All adverse events were non-serious injection site reactions of CTCAE Grade 1.

[0060] Example 3: Long-term stability testing of formulation candidates A long-term study was conducted at 50 mg / mL to observe the stability of etanercept in several new formulation candidates. Stability was assessed for 1 mL fills in 1 mL staked glass needle syringes using SE-HPLC, HIC HPLC, dSEC HPLC, and particulate analysis (HIAC) after storage at 4°C, 25°C, and 40°C. Osmolality and protein concentration were tested only at time zero, and pH was tested at time zero and after 12 weeks of storage to ensure there was no pH drift. The results of the study showed that the formulations tested maintained similarity to the current marketed formulation after 12 weeks at an accelerated temperature of 40°C and after 24 weeks at recommended storage at 2-8°C and an accelerated temperature of 25°C.

[0061] [Table 11]

[0062] Materials: PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) containing 50 mg / mL Enbrel drug substance was used in this study. The 50 mg / mL material was diafiltered into PASS and SAS_100NaCl (25 mM L-arginine, 100 mM NaCl, 1% sucrose) and then ultrafiltered to approximately 75 mg / mL. 50 mg / mL formulations were prepared by diluting the PASS and SAS material after UF / DF with the corresponding solutions. SAST_120NaCl was prepared by diluting the 75 mg / mL SAS material with a concentrated NaCl stock solution to a final concentration of 120 mM NaCl. A 1% stock solution of polysorbate 20 was freshly prepared and added to all formulations to a final concentration of 0.010%. All formulations were manually filled into 1 mL long BD glass syringes to a volume of 1 mL and then stoppered using an ASPU vacuum stopper device.

[0063] Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler Inlab MicroProbe. Samples were warmed to room temperature before measurement. Protein concentration measurements by absorbance at 280 nM were performed on all samples at room temperature using a DropSense96 UV / Vis Lab Chip DS system. Each sample was measured without addition, with at least three replicates (3 μL each), including a formulation solution blank. Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 μL of sample, and a 290 mOsm osmolality standard was tested to ensure the system was operating properly. Size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software. Hydrophobic interaction HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software at absorbance of 215 nm. Denaturing size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software. Subvisible particle analysis was performed using a HACH HIAC / Royco particle counter system equipped with an HRLD-150 laser and Pharm Spec software. All samples were diluted to 25 mg / mL with PASS formulation buffer. Samples were thoroughly mixed, uncapped, and degassed at 75 torr for 2 hours before analysis. Four replicate analyses of 1.0 mL each (no tare volume) were performed; the first analysis was discarded, and the remaining three analyses were averaged. Particle size data for 2, 5, 10, and 25 μm were collected at all time points. Results reflect dilution and are reported as cumulative counts per milliliter.

[0064] Results and Discussion: The pH of all formulations was measured at time zero and after 12 weeks at all temperatures. No trends were observed as a function of time or storage temperature. The measured pH values ​​for all samples are shown in Table 12. No pH drift was observed after 52 weeks of storage at 4°C, 24 weeks at 25°C, or 12 weeks at 40°C; all samples met the acceptance criteria of + / - 0.2 pH units from the target pH of 6.3.

[0065] [Table 12]

[0066] All formulations were tested for protein concentration at time zero. Protein concentration results for all samples are shown in Table 13. All samples met the acceptance criteria.

[0067] [Table 13]

[0068] Osmolality was tested only at time zero. The osmolality results for all samples are shown in Table 14. All formulations were at their target osmolality. Due to differences in buffer and excipient concentrations, osmolality was not expected to be the same between the various formulations.

[0069] [Table 14]

[0070] SE-HPLC was performed to observe aggregation levels as a function of formulation conditions, time, and temperature. Peak B is the amount of high molecular weight species (aggregates) formed. Results showed no difference in Peak B between the PASST control and the buffer-free formulation at 4°C and 25°C, with only slight differences observed after 12 weeks at 40°C (Figure 1). Peak B represents the total aggregates detected by SE-HPLC for these formulations. After 52 weeks of storage at 4°C, 24 weeks at 25°C, and 12 weeks at 40°C, all samples remained within acceptable limits (Peak B ≤ 6%).

[0071] Modified SE-HPLC was used to measure the HMW species and LMW. Results showed similar trends for HMW species, main peak, and LMW between formulations after 52 weeks (Figure 2).

[0072] Changes in misfolded aggregates were monitored by HIC HPLC. Results at all temperatures tested showed no differences in peak 3 between the PASST control and the buffer-free formulation (Figure 3). After 52 weeks of storage at 4°C, 24 weeks at 25°C, and 12 weeks at 40°C, all samples remained within acceptable limits (Peak 1 ≤ 5%, Peak 2 ≥ 70%, Peak 3 ≤ 28%).

[0073] Subvisible particles were measured by light obscuration particle counting (HIAC). Results were consistent with previous PFS data, with similar results across formulations at all temperatures after 12 weeks. Because a single vial containing three pooled syringes was used for each time point, and there was a high level of syringe-to-syringe variability in silicone oil droplet contribution, trends could not be established from this data set.

[0074] Conclusions: The long-term stability of several new candidate modifications and the current commercial formulation with added polysorbates was evaluated at 4°C, 25°C, and 40°C. No significant differences were observed between the formulations after 52 weeks at 4°C and 24 weeks at 25°C by SE-, dSEC, or HIC HPLC analysis and by light obscuration, but slight differences were observed by HPLC analysis after 12 weeks at 40°C. No pH drift was observed, and all formulations remained within acceptable limits. The results of the study showed that the 50 mg / mL SAST_120NaCl and SAST_100NaCl formulations were stable after 12 weeks at the recommended storage temperature of 2°C to 8°C, similar to the current commercial formulation.

[0075] Example 4: Freeze / thaw and long-term stability of top modification candidates in stainless steel containers A freeze / thaw cycle study was conducted at 50 mg / mL to observe the stability of etanercept in three new formulation candidates: SAST_100NaCl (25 mM L-arginine, 100 mM NaCl, 1% sucrose, 0.010% polysorbate 20), SAS_120NaCl (25 mM L-arginine, 120 mM NaCl, 1% sucrose), and SAST_120NaCl (25 mM L-arginine, 120 mM NaCl, 1% sucrose, 0.010% polysorbate 20), compared with the current commercial formulation, PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose). The stability against aggregation when cycled from -30°C to 4°C in a 55 mL stainless steel freezing container was evaluated using SE-HPLC for up to five freeze / thaw cycles.

[0076] Additionally, a long-term study was conducted at 50 mg / mL to observe the stability of etanercept in a new formulation candidate. The formulation compared to the current marketed formulation, PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) was SAST_120NaCl (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose, 0.010% polysorbate 20). Stability when stored in 10 mL and 55 mL stainless steel cryocontainers was evaluated using SE-HPLC, HIC HPLC, dSEC HPLC, and particulate matter (HIAC). Storage temperatures and time points were -30°C for up to 36 months and 4°C for up to 12 months. Results from 52 weeks are presented herein.

[0077] Results: The pH of all formulations remained consistent at 52 weeks and through five freeze / thaw cycles.

[0078] [Table 15]

[0079] [Table 16]

[0080] There was a slight increase in >2 and >5 μm particles in the SAST_120NaCl formulation, but no trend was observed by HIAC for >10 μm particles. As shown in Figures 4, 5, and 6, no significant differences were observed between formulations by HIC, dSEC, or SEC. After exposure to five freeze-thaw cycles, no significant changes between formulations were observed by SEC. See Figure 7.

[0081] Conclusions: The results of the study to date have shown that the new formulation tested remains similar to the current commercial formulation after 52 weeks of storage in stainless steel freezer containers at -30°C and 4°C.

[0082] Example 5: Exchange to SAS and PASS solutions The purpose of these examples was to dialyze different preparations of etanercept in TMS (Tris, mannitol, sucrose) into a test formulation (L-arginine, sucrose, NaCl) and compare the final pH to the target pH.

[0083] Materials: Etanercept: 25 mg / mL in TMS (10 mM Tris-HCl, 4% mannitol, 1% sucrose, pH 7.4); SAS_100NaCl solution for dialysis (100 mM NaCl, 25 mM L-arginine HCl, 1% sucrose, pH 6.3); PASS buffer (25 mM phosphate, 100 mM NaCl, 25 mM L-arginine HCl, 1% sucrose, pH 6.3); 10,000 MWCO centriprep; 3-12 mL Slide-A-Lyzer dialysis cassette, 10,000 MWCO; Mettler Toledo MP220 pH meter, and Mettler Toledo InLab MicroProbe.

[0084] Methods: For example, a 25 mg / mL etanercept solution in TMS was concentrated to approximately 50 mg / mL by ultrafiltration using a Millipore Pellicon-2 mini system with a 30K MWCO Pellicon 3 cassette. This material was then diafiltered against SAS_100NaCl or PASS solution at 7 diavolumes, followed by ultrafiltration to 100 mg / mL. For example, a 10,000 MWCO centriprep was used to concentrate a 25 mg / mL etanercept solution in TMS to 50 mg / mL. As with the SAS dialysis solution, the pH of the 50 mg / mL sample in TMS was measured using a Mettler Toledo MP220 pH meter and an InLab MicroProbe. This material was then dialyzed using a 10,000 MWCO slide-a-lyzer dialysis cassette. 9.5 mL of 50 mg / mL etanercept in TMS was added to the cassette and exchanged with 1000 mL of SAS100. Three exchanges were performed to achieve a 1,000,000-fold exchange. The first exchange occurred at 5:00 PM on Day 1 and continued overnight. The second 1,000 mL exchange occurred at 8:30 AM on Day 2. The third and final exchange occurred at 12:30 PM on Day 2. At 5:00 PM on Day 2, the protein was removed from the dialysis cassette (11 mL was removed) and the pH was measured, also using a Mettler Toledo MP220 pH meter. The measured pH was 6.98.

[0085] result: A summary of the results is shown in Table 17 below.

[0086] [Table 17]

[0087] Conclusion: When the sample was ultrafiltered / diafiltered into PASS buffer from a pre-exchange solution of pH 7.56, the target pH of 6.34 was achieved. However, when the sample was ultrafiltered / diafiltered into SAS_100NaCl solution, the pH of the post-dialysis material achieved was 6.98, which was higher than expected and not close to the final target pH of 6.3. The same results were obtained when using dialysis as the exchange method into SAS_100NaCl.

[0088] Example 6: UF / DF Pool Introduction: The formulation solution selected for this study was designated SAS (120 mM sodium chloride, 25 mM L-arginine, 1% sucrose, pH 6.3) and contained no added phosphate buffer. Previous examples demonstrated that starting with etanercept in a pH 7.56 sample, achieving a target pH of 6.3 was difficult, either by dialysis or using UF / DF, necessitating a different method of conversion to the SAS formulation. Two methods utilizing different final UF / DF starting materials were evaluated: 1) Column 3 (AEX) intermediate pool as the starting material, and 2) Enbrel drug substance in PASS formulation buffer (PASS DS intermediate pool) as the starting material. Each method is described below, summarizing the development of the final UF / DF unit operation process to produce 50 g / L of SAS-formulated etanercept, including preparation of the SAS formulation solution, and final UF / DF loading and processing.

[0089] Methods: The SAS formulation solution consisted of 120 mM sodium chloride, 25 mM L-arginine, 1% sucrose, pH 6.3. The SAS formulation solution was titrated to pH 6.3 using 10 N NaOH. The volume of titrant required to reach the specified pH range was 4.4 μL / L of SAS formulation solution. During the SAS final UF / DF unit operation, the membrane was immersed in 10 L / m of SAS formulation solution. 2After equilibration with HCl, the pH of the permeate remained closer to the pH of WFI than to the pH of the SAS formulation solution. Without being bound by theory, this is believed to be due to the low buffering capacity of the SAS formulation solution. The conductivity range of the permeate after membrane equilibration, predicted from the range of SAS formulation solution preparations, is 12-16 mS / cm. A higher pH after equilibration than the pH of the SAS formulation solution is expected and should not be a cause for concern or an indication that the membrane is not equilibrated.

[0090] AEX intermediate pool starting material: Before transferring the AEX intermediate pool to the retentate tank of the UF / DF tank, the pool was adjusted to a target pH of 6.3 (acceptable range 6.2-6.4) using 2 M HCl. The volume of titrant required to reach the specified pH range was approximately 2.8 mL / L of AEX intermediate pool.

[0091] Table 18 lists eight examples conducted during the development of a SAS final UF / DF unit operation process using an AEX intermediate pool as the starting material. Two parameters were investigated: pH of the adjusted AEX intermediate pool and pH of the SAS formulation solution. The first three experiments were analyzed for pH, conductivity, osmolality, protein concentration, and product quality. Experiments 4 through 7 measured only pH, conductivity, osmolality, and protein concentration to determine the effect of formulation solution pH and the effect of loading pH on the pH of the UF / DF pool.

[0092] [Table 18]

[0093] Results: Product quality results for the final SAS UF / DF pools produced using the AEX intermediate pool as the starting material are shown in Table 19. While the process yield for Run 1 was outside the acceptance criteria, this was most likely an artifact of bench-scale processing and was not considered critical to the conclusions of the study. As noted above, all three final UF / DF SAS runs also met the acceptance criteria for product quality using SEC and HIC analysis.

[0094] [Table 19]

[0095] Stability of the regulatory AEX intermediate pool The prepared AEX intermediate pool can be stored at controlled room temperature (CRT) for up to 52.6 hours. The pH of the pool during storage is shown in Figure 8.

[0096] Stability of UF / DF pool The final UF / DF SAS pool produced using the AEX intermediate pool as the starting material can be stored at CRT for up to 96.3 hours. The pH and conductivity during storage are shown in Figures 9A and B. The pH and conductivity remain within acceptable ranges during the 96.3 hour storage period.

[0097] PASS DS Intermediate Pool Starting Material: Because the PASS DS Intermediate Pool is already within an acceptable pH range, no adjustment is required before transferring the PASS DS Intermediate Pool to the UF / DF retentate tank. Additionally, the starting material is 50 mg / mL PASS formulated Enbrel DS, which is already the correct concentration for diafiltration, so there is no need to concentrate the pool to 50 g / L.

[0098] To evaluate the source of starting material, an example conducted during the development of a SAS final UF / DF unit operation process is shown in Table 20. This example utilized the DS PASS intermediate pool as the starting material and was analyzed for pH, conductivity, osmolality, protein concentration, and product quality.

[0099] [Table 20]

[0100] Results: Product quality results for the final SAS UF / DF pool produced using the PASS DS intermediate pool as the starting material are shown in Table 21. While the process yield for Run 1 was outside the acceptance criteria, this was most likely an artifact of bench-scale processing and was not considered critical to the conclusions of the study. As noted above, the final SAS UF / DF pool also met the acceptance criteria for product quality using SEC and HIC analysis.

[0101] [Table 21]

[0102] Stability of the PASS DS intermediate pool Because this intermediate pool is already at the target pH (6.3), no adjustment of this PASS pool is necessary prior to UF / DF treatment with SAS solution. Pool storage studies were not performed on this intermediate pool because the pool condition was not changed from Enbrel PASS DS. Pools can be stored at 25°C for up to 96 hours.

[0103] Stability of UF / DF pool The final UF / DF SAS pool, produced starting from the PASS DS intermediate pool, can be stored at CRT for up to 96.3 hours. The pH and conductivity during storage are shown in Figures 9A and B. The pH and conductivity remain within acceptable ranges throughout the 96.3 hour storage period.

[0104] Stability of SAS formulation solution The SAS formulation solution can be stored at CRT for up to 28 days. pH and conductivity are shown in Figure 10A and B. When stored for 42 days in a small-scale stainless steel stability chamber with a very small headspace, the SAS formulation solution was shown to maintain a pH between 5.6 and 6.5. Precipitation was observed at days 35 and 42. The 5.09 reading at day 21 appears to be an outlier due to the fact that the subsequent time points are within the proposed acceptance criteria.

[0105] Conclusions: The final UF / DF unit operation is capable of producing 50 g / L SAS-formulated product and can achieve consistent product quality compared to the currently commercially available PASS-formulated product under the following process recommendations: 1) utilize the AEX intermediate pool or PASS DS intermediate pool as the starting material; 2) the SAS solution can be stored at CRT for at least 28 days, maintaining a pH of 5.6-6.5; 3) the adjusted AEX intermediate pool can be stored at CRT for at least 52.6 hours, maintaining a pH of 6.3 ± 0.1; and 4) the SAS-formulated UF / DF pool can be stored at CRT for at least 96.3 hours, maintaining a pH of 6.1-6.5 and a conductivity of 10-14 mS / cmS.

[0106] Example 7: Isotonic replacement formulation The purpose of this example was to determine the effect of increasing concentrations of arginine, sucrose, or sodium chloride on etanercept stability at 75 mg / mL at 40°C, relative to aggregation. The concentrations of each of these excipients were increased to maintain an isotonic formulation without the addition of phosphate buffer. Additionally, histidine was evaluated as a phosphate replacement buffer. The formulations tested are summarized in Table 22.

[0107] [Table 22]

[0108] Material: Enbrel drug substance at 50 mg / mL in PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) was used in this study. The material was dialyzed into a new formulation (no polysorbate) and concentrated to a 75 mg / mL use using a 30,000 MWCO centriprep. A 1% stock solution of polysorbate 20 was freshly prepared and added to all formulations to a final concentration of 0.01%. All formulations were manually filled into 1 mL long BD glass syringes to a volume of 1.0 mL and then stoppered using an ASPU vacuum stopper device.

[0109] Methods: pH was measured using a Mettler Toledo pH meter in combination with a Mettler MicroProbe. Samples were warmed to room temperature before measurement. Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 μL of sample, and a 290 osmolality standard was tested to ensure the system was operating properly. Size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software.

[0110] Results: Concentrations, pH, and osmolality are shown in Table 23. The aggregation rates at 40°C and 75 mg / mL for all phosphate-free formulations were similar to the commercial formulation compositions, with increasing concentrations of L-arginine, sucrose, and NaCl, as shown in Table 24. Furthermore, the use of histidine instead of phosphate as a buffer increased the aggregation rate at 40°C.

[0111] [Table 23]

[0112] [Table 24]

[0113] Example 8: Stability of formulations with various concentrations of polysorbate 20 A long-term study was conducted to observe the stability of 50 mg / mL etanercept at 0, 0.005, 0.01, and 0.015% polysorbate 20 in the SAST formulation. Additionally, a 100 mg / mL SAST high-concentration formulation of etanercept was tested. After storage at 4°C, 25°C, and 40°C, stability was assessed using SE-HPLC, dSEC HPLC, and high-intensity artificial chromatographic analysis (HIAC) for 1 mL fills in 1 mL staked-needle syringes. Osmolality, pH, and protein concentration were tested only at time zero. Results indicated that the 50 mg / mL formulation tested maintained similarity to the current marketed formulation after 24 weeks at the recommended 2-8°C and accelerated temperatures of 25°C and 40°C. The 100 mg / mL SAST formulation behaved similarly to the 50 mg / mL formulation with respect to pH and subvisible particles; the difference in aggregation levels by SEC was attributed to protein concentration.

[0114] [Table 25]

[0115] Materials: 25 mg / mL Enbrel drug substance in TMS (10 mM Tris buffer, 4% mannitol, 1% sucrose) was used in this study. The bulk used for the SAS formulation was titrated to pH 6.3. The material was ultrafiltered to approximately 50 mg / mL etanercept and then diafiltered against PASS (25 mM phosphate, 25 mM L-arginine, 120 mM NaCl, 1% sucrose) or SAS (25 mM L-arginine, 120 mM NaCl, 1% sucrose) with 50 mg / mL etanercept. The material for the high concentration group was then ultrafiltered to 100 mg / mL etanercept. A 1% stock solution of polysorbate 20 was freshly prepared and added to the formulation to the final concentrations shown in Table 25. All formulations were manually filled into 1 mL long BD glass syringes to a volume of 1 mL and then stoppered using an ASPU vacuum stopper device.

[0116] Results and Discussion: The pH of all formulations was measured at time zero, after 12 weeks at 40°C, and after 24 weeks at 4°C and 25°C. No trends were observed as a function of time or storage temperature. The measured pH for all samples is shown in Table 26. No pH drift was observed after 12 weeks of storage at 40°C or 24 weeks of storage at 4°C and 25°C, and all samples met the acceptance criteria of a target pH of 6.3 ± 0.2 pH units. The protein concentration and osmolality of all formulations were tested at time zero. The protein concentration and osmolality results for all samples are shown in Table 26.

[0117] [Table 26]

[0118] SE-HPLC was performed to observe aggregation levels as a function of formulation conditions, time, and temperature. Peak B is the amount of high molecular weight species (aggregates) formed. Results showed no difference in Peak B between the PASS control and buffer-free formulations at all temperatures and each protein concentration (Tables 27-29). Peak B represents the total aggregates detected by SE-HPLC in these formulations. After 24 weeks of storage at 4°C and 25°C, and after 2 weeks of storage at 40°C, all 50 mg / mL samples remained acceptable (Peak B ≤ 6%).

[0119] Subvisible particles were measured by light obscuration particle counting (HIAC). Results were consistent with previous PFS data and were similar between formulations across all temperatures after 24 weeks (Table 30).

[0120] [Table 27]

[0121] [Table 28]

[0122] [Table 29]

[0123] [Table 30]

[0124] Conclusions: The long-term stability of candidate and current marketed formulations of 50 mg / mL etanercept with polysorbate concentrations ranging from 0 to 0.015% was evaluated at 4°C, 25°C, and 40°C. A higher concentration of 100 mg / mL etanercept was also tested for the SAS010T formulation. No significant differences were observed between formulations of each protein concentration after 24 weeks by SE-, dSEC, or HIC HPLC analysis and light obscuration. No pH drift was observed, and all formulations remained within acceptable limits. The results of the study showed that the 50 mg / mL SAST_120NaCl formulation was stable at recommended storage temperatures of 2°C to 8°C after 24 weeks, similar to the current marketed formulation.

[0125] Example 9: Stability of formulations in plastic syringes A long-term study was conducted to observe the stability of etanercept in PASS and SAS formulations of 50 mg / mL etanercept in COP plastic silicone oil-free prefilled syringe systems compared to glass siliconized prefilled syringes. After storage at 4°C, 25°C, and 40°C, stability was assessed for 1 mL fills in the various syringe systems using SE-HPLC, pH, and particulate matter (HIAC). Protein concentration was tested only at time zero.

[0126] Materials: TMS (10 mM Tris buffer, 4% mannitol, 1% sucrose) containing etanercept drug substance at 25 mg / mL etanercept was used in this study. The bulk used for SAS formulation was titrated to pH 6.3. The material was ultrafiltered to approximately 50 mg / mL etanercept and then diafiltered against PASS (25 mM phosphate, 25 mM L-arginine, 120 mM NaCl, 1% sucrose) or SAS (25 mM L-arginine, 120 mM NaCl, 1% sucrose) at 50 mg / mL etanercept. All formulations were manually filled to a volume of 1 mL into 1 mL long BD glass syringes or 1 mL COP plastic silicone oil-free syringes (COP_A and COP_B) and then stoppered using a vacuum stopper device.

[0127] Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler Inlab MicroProbe. Samples were warmed to room temperature before measurement. Protein concentration measurements for all samples were performed at room temperature using the Nano Drop system, using absorbance at 280 nM. Size-exclusion HPLC was performed using an Agilent 1100 HPLC with Chromeleon 7.2 software. Subvisible particle analysis was performed using a HACH HIAC / Royco particle counter system equipped with an HRLD-150 laser and Pharm Spec software. All samples were diluted to 25 mg / mL with PASS formulation buffer. Samples were thoroughly mixed, uncapped, and degassed at 75 torr for 2 hours before analysis. Four replicate analyses of 1.0 mL each (no tare volume) were performed; the first analysis was discarded, and the remaining three analyses were averaged. Particle size data for 2, 5, 10, and 25 μm were collected at all time points. Results reflect dilution and are reported as cumulative counts per mL.

[0128] Results and Discussion: Stability in plastic silicone oil-free syringes is similar to stability in glass siliconized syringes. Protein concentration of all formulations was tested at time zero. pH of all formulations was measured at time zero, after 12 weeks at 40°C, and after 24 weeks at 4°C and 25°C. No trends were observed as a function of time or storage temperature, and all samples met the acceptance criteria of a target pH of 6.3 ± 0.2 pH units. Protein concentration and pH measurements for all samples are shown in Table 31.

[0129] [Table 31]

[0130] SE-HPLC was performed to observe aggregation levels as a function of formulation conditions, time, and temperature. Peak B is the amount of high molecular weight species (aggregates) formed. Results showed no difference in Peak B between glass syringes and COP plastic syringes without silicone oil (Tables 32-34). Peak B represents the total aggregates detected by SE-HPLC in these formulations. After 24 weeks of storage at 4°C and 25°C, all samples remained acceptable (Peak B ≤ 6%).

[0131] [Table 32]

[0132] [Table 33]

[0133] [Table 34]

[0134] Subvisible particles were measured by light obscuration particle counting (HIAC). Results for formulations filled in BD glass syringes were consistent with previous PFS data, but subvisible particles were reduced in plastic syringes without silicone oil (Table 35).

[0135] [Table 35]

[0136] Conclusions: The long-term stability of a 50 mg / mL etanercept SAS formulation and the current marketed etanercept formulation stored in glass siliconized syringes and syringes without COP silicone oil was evaluated at 4°C, 25°C, and 40°C. No significant differences were observed between formulations as a function of syringe type by SE-HPLC after 24 weeks. No pH drift was observed, and all formulations remained within acceptable limits. Subvisible particles were reduced in plastic syringes without COP silicone oil, consistent with previous PFS results when stored in glass syringes. The results of the study showed that the 50 mg / mL etanercept SAS formulation was stable in various syringes at recommended storage temperatures of 2°C to 8°C after 24 weeks, similar to the current marketed formulation.

Claims

1. 1. A pharmaceutical composition comprising 75 mM to 150 mM NaCl, 5 mM to 100 mM arginine, 0.5% to 2% (w / v) sucrose, and 40 mg / mL to 100 mg / mL etanercept, wherein the pharmaceutical composition contains less than 2.0 mM total added buffering agents, and the pH of the composition is 6.1 to 6.

5.

2. 10. The pharmaceutical composition of claim 1, comprising less than 1.5 mM total additional buffering agent.

3. 3. The pharmaceutical composition of claim 2, comprising less than 1.0 mM total additional buffering agent.

4. 3. The pharmaceutical composition of claim 2, comprising less than 0.5 mM total additional buffering agent.

5. 3. The pharmaceutical composition of claim 2, comprising less than 0.25 mM total additional buffering agent.

6. 3. The pharmaceutical composition of claim 2, comprising no more than 0.1 mM total additional buffering agent.

7. 2. The pharmaceutical composition of claim 1, wherein the arginine is L-arginine.

8. 8. The pharmaceutical composition of claim 7, wherein the L-arginine is L-arginine hydrochloride.

9. 8. The pharmaceutical composition of claim 7, wherein the L-arginine is L-arginine base.

10. 10. The pharmaceutical composition of claim 1, which maintains a pH of 6.1 to 6.5 when stored at controlled room temperature (CRT) for two weeks.

11. 11. The pharmaceutical composition of claim 10, which maintains a pH of about 6.2 to about 6.3 when stored at controlled room temperature (CRT) for two weeks.

12. 10. The pharmaceutical composition of claim 1, wherein when stored at about 25° C., the composition maintains a pH of 5.8 to 6.7 for at least two weeks, and wherein less than 6% of the total etanercept aggregates in a high molecular weight form as assessed using size exclusion chromatography.

13. 10. The pharmaceutical composition of claim 1, wherein the composition maintains a pH of about 6.1 to about 6.5 during said storage at about 25°C for at least two weeks.

14. 10. The pharmaceutical composition of claim 1, wherein the composition maintains a pH of about 6.2 to about 6.4 during said storage at about 25°C for at least two weeks.

15. 10. The pharmaceutical composition of claim 1, wherein after 2 weeks of storage at about 25°C, less than 28% of the total amount of etanercept is in misfolded form as assessed using hydrophobic interaction chromatography.

16. 10. The pharmaceutical composition of claim 1, having an osmolality of about 180 to about 420 millimolar osmolality.

17. 17. The pharmaceutical composition of claim 16, having an osmolality of about 250 to about 350 millimolar osmolality.

18. 18. The pharmaceutical composition of claim 17, having an osmolality of about 290 to about 310 millimolar osmolality.

19. 19. The pharmaceutical composition of claim 18, having an osmolality of about 300 to about 310 millimolar osmolality.

20. 2. The pharmaceutical composition of claim 1, consisting essentially of about 50 mg / mL etanercept, about 120 mM NaCl, about 25 mM L-arginine hydrochloride, about 1% (w / v) sucrose, and water.

21. 10. The pharmaceutical composition of claim 1, consisting of about 50 mg / mL etanercept, about 120 mM NaCl, about 25 mM L-arginine hydrochloride, about 1% (w / v) sucrose, and water.

22. 10. The pharmaceutical composition of claim 1, further comprising polysorbate 20.

23. 23. The pharmaceutical composition of claim 22, wherein the concentration (w / v) of polysorbate 20 is from about 0.001% to about 0.1%.

24. 24. The pharmaceutical composition of claim 23, wherein the concentration (w / v) of polysorbate 20 is about 0.005%, about 0.01%, or about 0.015%.

25. 1. A method for producing an etanercept pharmaceutical composition that does not contain an additional buffering agent and has a pH of 6.1 to 6.5, the method comprising the step of exchanging an etanercept formulation that contains an additional buffering agent with a solution that does not contain an additional buffering agent, the resulting pharmaceutical composition comprising 40 mg / mL to 100 mg / mL of etanercept, and the etanercept formulation with an additional buffering agent and the solution without an additional buffering agent each having a pH of 6.1 to 6.

5.

26. 26. The method of claim 25, wherein the exchanging step uses diafiltration.

27. 26. The method of claim 25, wherein the solution without added buffer is isotonic.

28. 26. The method of claim 25, wherein the solution without additional buffering agents contains sucrose, arginine, and NaCl.

29. 29. The method of claim 28, wherein the solution without additional buffering agents contains 75 mM to 150 mM NaCl, 5 mM to 100 mM arginine, and 0.5% to 2% (w / v) sucrose.

30. 30. The method of claim 28, wherein the solution without additional buffer consists essentially of about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water.

31. 26. The method of claim 25, further comprising filtering the pharmaceutical composition.

32. 26. The method of claim 25, further comprising aliquoting the pharmaceutical composition into drug product forms.

33. 26. The method of claim 25, wherein the etanercept formulation with an additional buffering agent comprises 75 mM to 150 mM NaCl, the solution without an additional buffering agent comprises 75 mM to 150 mM NaCl, the pharmaceutical composition comprises 75 mM to 150 mM NaCl, and the method does not include a NaCl removal step.

34. 34. The method of claim 33, wherein the etanercept formulation with additional buffering agent comprises about 120 mM NaCl, the solution without additional buffering agent comprises about 120 mM NaCl, and the pharmaceutical composition comprises about 120 mM NaCl.

35. 26. The method of claim 25, which does not include a salt removal step.

36. 26. A pharmaceutical composition comprising 75 mM to 150 mM NaCl, 5 mM to 100 mM arginine, 0.5% to 2% (w / v) sucrose, and 40 mg / mL to 100 mg / mL etanercept, comprising less than 2.0 mM total additional buffering agents, and wherein the pH of the composition is 6.1 to 6.5, and wherein the pharmaceutical composition is produced using the method of claim 25.

37. A kit comprising the pharmaceutical composition of claim 1 in pharmaceutical product form and instructions for storage and use.

38. A single-dose container containing the pharmaceutical composition of claim 1.

39. 39. The single-dose container of claim 38, which is a vial, syringe, or autoinjector.

40. 39. The single-dose container of claim 38, comprising an aqueous formulation consisting of 50.0 mg / mL etanercept, 120 mM sodium chloride, 25 mM L-arginine hydrochloride, and 1.0% (w / v) sucrose.

41. 10. A method for preparing a single-dose container, comprising filling the single-dose container under aseptic conditions with approximately a single dose of the pharmaceutical composition of claim 1.

42. 10. A method of treating a patient with rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or psoriasis, comprising administering to said patient the pharmaceutical formulation of claim 1.

Images