EA041785B1 - PHARMACEUTICAL COMPOSITION AND METHOD FOR ITS OBTAINING - Google Patents

Description

Cross-reference to related application

The application for this invention claims the priority of U.S. Provisional Application No. 62/411458, filed October 21, 2016, which is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to formulations of pharmaceutical compositions based on etanercept. In addition, the invention relates to methods for debuffering and formulation of pharmaceutical compositions based on etanercept.

Background of the invention

A protein drug formulation can present many challenges to the pharmaceutical scientist. There is a need for a formulation that stabilizes a protein drug and renders it stable against degradation due to proteolysis, aggregation, misfolding, and the like. In particular, for engineered proteins that differ substantially from known proteins, establishing suitable stability conditions can be a challenge. It is also desirable that the protein drug be in a form convenient for the patient. Required properties include stability at room temperature and when cooled; suitability for long-term storage, appropriate dosing frequency and volume, and minimization of discomfort upon administration.

Etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the 75 kD human tumor necrosis factor receptor (TNFR) (p75) linked to the Fc portion of human IgG1. The Fc component of etanercept contains a CH2 domain, a CH3 domain and a hinge region, but does not contain an IgG1 CH1 domain. When expressed in mammalian cells, it forms a homodimeric complex with two TNF receptor domains. Thus, it is an artificial protein that differs from both antibodies and soluble TNF receptors, and therefore undergoes different degradation pathways than either. Etanercept is commercially available under the brand name ENBREL® (Amgen Inc., Thousand Oaks, Calif.) 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 or older, moderate to severe chronic plaque psoriasis (PsO) in adults, psoriatic arthritis (PsA) in adults, and active ankylosing spondylitis (AS). Etanercept was initially available as a lyophilized formulation for reconstitution immediately prior to injection.

After reconstitution of the lyophilized product with water for injection, the formulation contains 10 mM Tris-HCl, 4% mannitol, 1% sucrose, pH 7.4 at a concentration of approximately 25 mg/ml. However, this formulation is not storage stable. It has been found that an etanercept liquid formulation can be prepared using arginine to stabilize the protein (see US Pat. No. 7,648,702). An exemplary liquid formulation consists of 50 mg/ml etanercept, 25 mM phosphate buffer, 25 mM L-arginine hydrochloride, 100 mM NaCl, 1% sucrose at pH 6.3 in water.

Brief description of the invention

This document provides new and improved formulations based on etanercept. In particular, the present invention provides pharmaceutical compositions containing etanercept that are stable and conveniently stored as a liquid at controlled room temperature (CRT) for extended periods of time, even in the absence of an additional buffering agent. In addition, when the pharmaceutical compositions of the present invention are administered to subjects, they also exhibit significantly reduced pain upon injection compared to a commercially available formulation of the prior art. Thus, these pharmaceutical compositions are more convenient and preferred by patients.

In another aspect of the present invention, methods are provided for formulating etanercept pharmaceutical formulations at the desired pH, but in the absence of an additional buffering agent in the final formulation.

In another aspect, the present invention provides a pharmaceutical composition comprising etanercept, NaCl, arginine and sucrose, wherein the pharmaceutical composition is substantially free of additional buffering agent and the pH of the composition is between 6.1 and 6.5. In one embodiment, the pharmaceutical composition is capable of maintaining a pH in the range of 6.1 to 6.5 when stored at controlled room temperature (CRT) for 2 weeks. In another embodiment, the concentration of etanercept is 40 to 100 mg/mL. In another embodiment, the pharmaceutical composition is isotonic. In another embodiment, the pharmaceutical composition contains 20 to 150 mM NaCl, 5 to 100 mM arginine, and 0.5 to 2% (w/v) sucrose. In another embodiment, the pharmaceutical composition contains 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 (w/v) of 0.001 to 0.1 %. In another embodiment, the surfactant is polysorbate 80 at a concentration (w/v) of 0.001 to 0.1%. In another embodiment, the surfactant is poloxamer 188 at a concentration (w/v) of 0.01 to 0.3%. In another embodiment, the pH of the pharmaceutical composition is maintained in the range of 5.8 to 6.7 for at least two weeks when stored at approximately 25°C, where less than 6% of the total etanercept is aggregated in high molecular weight form, as determined with using size exclusion chromatography. In another embodiment, the pH of the pharmaceutical composition is maintained in the range of about 6.1 to about 6.5. In another embodiment, less than 28% of the total etanercept is in misfolded form as determined using hydrophobic interaction chromatography. In another embodiment, the pharmaceutical composition essentially consists of and about 40-100 mg/ml etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose and water. In another embodiment, the pharmaceutical composition essentially consists 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.

In another aspect, the present invention provides a method of formulating an etanercept-based pharmaceutical composition while removing the additional buffering agent and maintaining a pH in the range of 6.1 to 6.5, comprising formulating the etanercept-based pharmaceutical composition in a composition containing an additional buffering agent at a pH of from 6.1 to 6.5, and replacing the composition containing an additional buffer agent against a composition that does not contain an additional buffer agent and is characterized by a pH of 5.6 to 6.5, and collecting the resulting pharmaceutical composition. In one embodiment, diafiltration is used in the replacement step. In another embodiment, the composition, which does not contain additional buffering agent, is isotonic. In another embodiment, the composition, which does not contain additional buffering agent, contains sucrose, arginine and NaCl. In another embodiment, the formulation, which does not contain additional buffering agent, contains 20 to 150 mM NaCl, 5 to 100 mM arginine, and 0.5 to 2% (w/v) sucrose. In another embodiment, the composition, which does not contain additional buffering agent, essentially consists of about 120 mm NaCl, about 25 mm arginine, about 1% sucrose and water. In another embodiment, the method for formulating the etanercept pharmaceutical composition further comprises the addition of 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 for formulating an etanercept pharmaceutical composition further comprises filtering the pharmaceutical composition. In another embodiment, the method for formulating the etanercept pharmaceutical composition further comprises dividing the pharmaceutical composition into aliquots in the form of a finished dosage form.

In another aspect, the present invention provides a kit containing the etanercept pharmaceutical composition described above in the form of a finished dosage form and instructions for storage and use.

In another aspect, the present invention provides a pharmaceutical composition comprising etanercept, NaCl, arginine, sucrose, phosphate buffer and benzyl alcohol, wherein the pH of the composition is between 6.1 and 6.5. In one embodiment, the benzyl alcohol has a concentration (v/v) of 0.1 to 5.0%. In another embodiment, the concentration of benzyl alcohol is approximately 0.9%. In another embodiment, the pharmaceutical composition containing etanercept further comprises polysorbate 20 at a concentration (w/v) of 0.001 to 0.1%. In another embodiment, the concentration of polysorbate 20 is approximately 0.004%. In another embodiment, a pharmaceutical composition containing etanercept essentially consists of etanercept at a concentration of approximately 40-100 mg/ml, arginine at a concentration of approximately 25 mM, sodium chloride at a concentration of approximately 100 mM, sucrose at a concentration of ( w/v) of approximately 1%, phosphate buffer at a concentration of approximately 25 mM, and benzyl alcohol at a concentration (v/v) of approximately 0.9%. In another embodiment, a pharmaceutical composition containing etanercept essentially consists of etanercept at a concentration of approximately 40-100 mg/ml, arginine at a concentration of approximately 25 mM, sodium chloride at a concentration of approximately 100 mM, sucrose at a concentration of ( w/v) of approximately 1%, phosphate buffer at a concentration of approximately 25 mM, benzyl alcohol at a concentration (v/v) of approximately 0.9%, and polysorbate 20 at a concentration (w/v). vol.), which is approximately 0.004%.

In another aspect, the present invention provides a single dose container containing

- 2 041785 the above-described pharmaceutical composition containing etanercept. In one embodiment, the pharmaceutical composition essentially consists of etanercept at a concentration of approximately 40-100 mg/mL, arginine at a concentration of approximately 25 mM, sodium chloride at a concentration of approximately 100 mM, sucrose at a concentration (w/v .), constituting approximately 1%, phosphate buffer at a concentration of approximately 25 mm, and benzyl alcohol at a concentration (v/v) of approximately 0.9%. In another embodiment, the pharmaceutical composition essentially consists of etanercept at a concentration of approximately 40-100 mg/mL, arginine at a concentration of approximately 25 mM, sodium chloride at a concentration of approximately 100 mM, sucrose at a concentration (w/v .) of approximately 1%, phosphate buffer at a concentration of approximately 25 mM, benzyl alcohol at a concentration (v/v) of approximately 0.9%, and polysorbate 20 at a concentration (w/v) amounting to approximately 0.004%. In another embodiment, the single dose container is a vial, syringe, or pen. In another embodiment, the single-dose container contains an aqueous formulation consisting of etanercept at a concentration of 50.0 mg/ml, sodium chloride at a concentration of 120 mM, L-arginine at a concentration of 25 mM, sucrose at a concentration of 1.0% (w/v ).

In another aspect, the present invention provides a method for preparing a single dose container containing an etanercept-containing pharmaceutical composition as described above, comprising filling the single dose container with approximately a single dose of the pharmaceutical composition under sterile conditions.

Brief description of graphic materials

In FIG. 1 shows the percentage of HMW (peak B) detected by the SEC performed to analyze the stability of etanercept in Example 3.

In FIG. 2 shows the percentage of LMW detected by dSEC conducted to analyze the stability of etanercept in Example 3.

In FIG. 3 shows the percentage of peak 3 detected by the HIC performed to analyze the stability of etanercept in Example 3.

In FIG. 4 shows the percentage of peak 3 detected by the HIC performed to analyze the storage stability of etanercept in the stainless steel cryovessel in Example 4.

In FIG. 5 shows the percentage of LMW detected by dSEC conducted to analyze the stability of etanercept when stored in the stainless steel cryovessel in Example 4.

In FIG. 6 shows the percentage of peak B detected by the SEC performed to analyze the storage stability of etanercept in the stainless steel cryovessel in Example 4.

In FIG. 7 shows the percentage of peak B detected by the SEC performed to analyze the freeze-thaw stability of etanercept in Example 4.

In FIG. 8 shows the pH stability of the conditioned AEX intermediate pool at controlled room temperature (CRT) in the assay of Example 6.

In FIG. 9 shows the stability of pH (A) and conductivity (B) of the UF/DF pool at CRT in the assay of Example 6.

In FIG. 10 shows the stability of etanercept formulated in SAS solution with respect to pH (A) and conductivity (B) in the assay of Example 6.

Detailed description of the invention

The present invention provides improved pharmaceutical compositions based on etanercept. As used herein, the term "pharmaceutical composition" is intended to refer to a polypeptide-based formulation suitable for injection and/or administration to a patient in need thereof. More specifically, the pharmaceutical composition is essentially sterile and does not contain any agents that are excessively toxic or pathogenic 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 under the tradename ENBREL® (Immunex Inc., Thousand Oaks, CA). Etanercept is produced using recombinant DNA technology in the mammalian cell expression system - Chinese Hamster Ovary (CHO). It consists of 934 amino acids and has an average molecular weight of approximately 150 kDa (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 in this sequence (up to 10%) are possible and can be used within the scope of the present invention.

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Leu-Pro-Ala-Gln-Val-Ala-Phe-Thr-Pro-Tyr-

Ala-Pro-Glu-Pro-Gly-Ser-Thr-Cys-Arg-Leu-

Arg-Glu-Tyr-Tyr-Asp-Gln-Thr-Ala-Gln-Met-

Cys-Cys-Ser-Lys-Cys-Ser-Pro-Gly-Gln-His-

Ala-Lys-Val-Phe-Cys-Thr-Lys-Thr-Ser-Asp-

Thr-Val-Cys-Asp-Ser-Cys-Glu-Asp-Ser-Thr-

Tyr-Thr-Gln-Leu-Trp-Asn-Trp-Val-Pro-Glu-

Cys-Leu-Ser-Cys-Gly-Ser-Arg-Cys-Ser-Ser-

Asp-Gln-Val-Glu-Thr-Gln-Ala-Cys-Thr-Arg91 Glu-Gln-Asn-Arg-I le-Cys-Thr-Cys -Arg-Pro101 Gly-Trp-Tyr-Cys-Ala-Leu -Ser-Lys-Gln-Glu111 Gly-Cys-Arg-Leu-Cys-Ala-Pro-Leu-Arg-Lys121 Cys-Arg-Pro-Gly-Phe-Gly-Val-Ala-Arg-Pro131 Gly-Thr- Glu-Thr-Ser-Asp-Val-Val-Cys-Lys141 Pro-Cys-Ala-Pro-Gly-Thr-Phe-Ser-Asn-Thr151 Thr-Ser-Ser-Thr-Asp-Ile-Cys-Arg- Pro-His161 Gln-Ile-Cys-Asn-Val-Val-Ala-Ile-Pro-Gly171 Asn-Ala-Ser-Met-Asp-Ala-Val-Cys-Thr-Ser181 Thr-Ser-Pro-Thr-Arg -Ser-Met-Ala-Pro-Gly191 Ala-Val-His-Leu-Pro-Gln-Pro-Val-Ser-Thr2 01 Arg-Ser-Gln-His-Thr-Gln-Pro-Thr-Pro-Glu211 Pro -Ser-Thr-Ala-Pro-Ser-Thr-Ser-Phe-Leu221 Leu-Pro-Met-Gly-Pro-Ser-Pro-Pro-Ala-Glu231 Gly-Ser-Thr-Gly-Asp-Glu-Pro -Lys-Ser-Cys241 Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro251 Al a-Pro-Glu-Leu-Leu-Gly-Gly-Pr o-Ser-Vai 2 61 Phe- Leu-Phe-Pro-Pro-Lys-Pro-Lys-Asp-Thr271 Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr2 81 Cy s-Vai-Vai-Vai-Asp-Val- Ser-Hi s-Glu-Asp2 91 Pro-Glu-Val-Lys-Phe-Asn-Trp-Tyr-Val-Asp301 Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys311 P ro-Arg-Glu-Glu-Gln-Tyr-Asn-Ser-Thr-Tyr321 Arg-Val-Val-Ser-Val-Leu-Thr-Val-Leu-His331 Gln-Asp-Trp-Leu-Asn-Gly- Lys-Glu-Tyr-Lys341 Cys-Lys-Val-Ser-Asn-Lys-Ala-Leu-Pro-Ala351 Pro-Ile-Glu-Lys-Thr-Ile-Ser-Lys-Ala-Lys-

61 Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-

371 Leu-Pro-Pro-Ser-Arg-Glu-Glu-Met-Thr-Lys381 Asn-Gln-Val-Ser-Leu-Thr-Cys-Leu-Val-Lys-

91 Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-

01 Trp-Glu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-

411 Tyr-Lys-Thr-Thr-Pro-Pro-Val-Leu-Asp-Ser421 Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Lys-Leu431 Thr-Val-Asp-Lys-Ser-Arg -Trp-Gln-Gln-Gly441 Asn-Val-Phe-Ser-Cys-Ser-Val-Met-His-Glu-

51 Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-

461 Leu-Ser-Leu-Ser-Pro-Gly-Lys

The present invention provides a pharmaceutical composition containing etanercept but substantially no additional buffering agent. The term additional buffering agent refers to a component of an etanercept composition or formulation, other than etanercept itself, that contributes significantly to the buffering capacity of the composition or formulation. Etanercept itself has been shown to provide all the necessary buffering activity to maintain a pH in the range of 6.1 to 6.5 and in particular about 6.2 to 6.3 under the conditions described below. As demonstrated in Example 1 below, this pH range has been shown to be effective in maintaining the desired stability characteristics of an etanercept formulation (less than 6% high molecular weight aggregates and less than 28% misfolded and truncated compounds).

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The expression essentially does not contain an additional buffer means that it contains less than 0.5 mm of any buffer, in addition to etanercept.

The expression general additional buffering agent collectively refers to all components of an etanercept composition or formulation, with the exception of etanercept itself, which contribute significantly to the buffering capacity of the composition or formulation.

In some embodiments, the pharmaceutical compositions of the present invention contain less than 2.0 mM total co-buffer, less than 1.5 mM total co-buffer, less than 1.0 mM total co-buffer, less than 0.5 mM total co-buffer, less than 0.25 mM total additional buffer, less than 0.1 mM total additional buffer, or less than 0.05 mM total additional buffer. In typical pharmaceutical compositions, additional buffering agents are often used at concentrations of 5.0 mM or higher to maintain the pH in the desired range. Various well known additional buffering agents are 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 its buffering capacity is at or near pH 6.2. Sodium phosphate is the buffering agent used in today's commercial etanercept liquid formulation because its required pH is 6.3. In the present invention described herein, the etanercept pharmaceutical formulation is substantially free of sodium phosphate. Surprisingly, despite the virtual absence of any additional buffering agent, the pH of the pharmaceutical composition of the present invention is maintained in the range of 6.1 to 6.5 even after long-term storage. Even more surprisingly, when administered to subjects (eg, a subject or human patient), the pharmaceutical composition essentially without additional buffering agent causes significantly less pain than the current buffered commercial composition. Although phosphate is often chosen as a buffer for pharmaceutical compositions because of its near-neutral pH buffering capacity and belief that it is one of the least painful buffer components (compared to, for example, citrate buffer), the authors of the present of the invention have determined that a phosphate buffer at a pH of about 6.3 increases pain upon injection.

Unless otherwise clear from the context of its use, a formulation solution or formulation buffer is a solution or buffer that does not actually contain etanercept but is used to prepare a formulation containing etanercept.

Typically, the concentration of etanercept in the pharmaceutical compositions of the present invention is from about 40 to about 200 mg/ml in an aqueous formulation (particularly water as solvent). More preferably, the concentration of etanercept is from about 40 to about 100 mg/ml, even more preferably from about 40 to about 75 mg/ml, and optionally about 50 mg/ml.

The pharmaceutical compositions of the present invention also contain arginine. Arginine has been shown to contribute significantly to the stability of etanercept in a liquid formulation (see US Pat. No. 7,648,702, incorporated herein by reference). Pharmaceutically suitable forms of arginine are commercially available. Typically, L-arginine (eg, L-arginine HCl or L-arginine in base form) is the arginine used for pharmaceutical formulations. It is understood that in the pH range of 6.0 to 6.6, and in particular at a pH of about 6.2-6.3, arginine does not significantly contribute to the buffering capacity of the formulation. Thus, 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 from about 1 mM to about 1 M, more preferably from about 10 to about 200 mM, or alternatively from about 5 to about 100 mM, more preferably from about 10 to about 100 mM, still more preferably from about 15 to about 75 mM, and even more preferably about 25 mM. Thus, in one aspect of the present invention, the pharmaceutical composition contains from about 50 to 75 mg/ml of etanercept and about 25 mM of arginine, where the pharmaceutical composition contains essentially no additional buffering agent, and the pH of the composition is from 6.0 to 6.6 . The term used herein should be roughly understood to mean that a change in the concentration of a component of the described composition is allowed, which can be up to 10% of the set value and include it. For example, if a formulation contains approximately 10 mg/mL of a polypeptide, this means that the formulation may contain from 9 to 11 mg/mL of the claimed polypeptide.

Pharmaceutical compositions may contain additional excipients, provided that these excipients are not additional buffering agents and in particular are not phosphate buffering agents. Examples of additional excipients of the present invention include, without limitation, sugars/polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueous solvents such as polyhydric alcohols (eg PEG, ethylene glycol and glycerol), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF); amino acids such as proline, L-serine, alanine, glycine, lysine hydrochloride, sarcosine, and gamma-aminobutyric acid; and surfactants.

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

The osmolality of the pharmaceutical composition is preferably controlled in order to maximize the stability of the active ingredient as well as to minimize patient discomfort upon administration. It is generally preferred that the pharmaceutical composition be isotonic with blood serum, i.e. had the same or similar osmolality as achieved by adding a tonicity modifier. Serum has an osmolality of about 300±50 mOsm/kg, so an isotonic pharmaceutical composition is expected to have an osmolality of about 180 to about 420 mOsm. In some embodiments, the range will be from about 250 to about 350 mOsm.

By tonicity modifier is meant a molecule that contributes to the osmolality of a solution. Examples of tonicity modifiers suitable for modifying osmolality include, without limitation, amino acids (eg, arginine, cysteine, histidine, and glycine), salts (eg, sodium chloride, potassium chloride, and sodium citrate), and/or saccharides (eg, sucrose, glucose and mannitol). The concentration of tonicity modifier in the formulation is preferably from about 1 mM to about 1 M, more preferably from about 10 to about 200 mM. In some embodiments, NaCl and sucrose concentrations are adjusted to produce a pharmaceutical composition that is isotonic. In some embodiments, illustrated below by way of example, the pharmaceutical composition contains about 40-100 mg/ml etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water. In particular, the pharmaceutical composition may essentially consist of about 50-100 mg/ml etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, about 0.01% polysorbate 20, and water.

Optionally, the pharmaceutical compositions of the present invention may include a surfactant. Surfactants are agents that reduce stress at the solution/surface interface. Examples of surfactants are 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 copolymer, poloxamers such as poloxamer 188 (eg, PLURONIC® F-68 (Sigma-Aldrich, St. Louis, MO) or poloxamer 407 (eg, 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 pharmaceutical compositions at a concentration (w/v) of about 0.001 to about 0.03% In the specific embodiments illustrated below by way of example, polysorbate 20 can be included in pharmaceutical formulations at a concentration (w/v) of 0.01% or about 0.004%.

Testing of pharmaceutical compositions.

The following examples illustrate how a person skilled in the art can determine if a formulation is capable of maintaining the pH in the desired range. In essence, the pharmaceutical composition is formulated and stored in test containers (which may be glass vials, glass syringes, plastic syringes, stainless steel vessels, or any kind of sterile device suitable for pharmaceutical compositions) and the pH is determined at time 0 and then at specified times, depending on the circumstances. Typically, testing conditions require storage of the pharmaceutical composition, and these conditions are particularly emphasized. For example, the compositions of the present invention are capable of maintaining the desired pH at controlled room temperature (CRT) for at least

- 6041785 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks and at least 24 weeks. CRT is defined according to the USP (United States Pharmacopeia) and has an incubated temperature that covers the normal and generally accepted standard operating environment in the range of 20 to 25°C (68 to 77°F); this gives an average calculated kinetic temperature of no more than 25°C and allows for the range of 15 to 30°C (59 to 86°F) that occurs in pharmacies, hospitals and warehouses.

In one aspect, the pharmaceutical compositions of the present invention exhibit certain qualitative characteristics. Testing of these qualities is also described as an example below. For example, the pharmaceutical compositions of the present invention contain less than 6% of total etanercept aggregated in high molecular weight form as determined using size exclusion chromatography. As another example, the pharmaceutical compositions of the present invention contain less than 28% of the total amount of etanercept in misfolded form as determined using hydrophobic interaction chromatography.

The pharmaceutical compositions of the present invention are able to remain stable by maintaining the pH and/or other specified quality characteristics (minimum high molecular weight forms and minimum misfolded forms) at the following temperatures and extended periods of time: (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) for 1 freeze/thaw cycle, up to 2 freeze/thaw cycles, up to 3 freeze/thaw cycles, and up to 5 freeze/thaw cycles; (3) at 4°C (refrigerator 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.

The pharmaceutical compositions of the present invention also show an unexpected result in reducing pain when injected into a subject. This property can be assessed using the Visual Analogue Scale (VAS), which was established by Gallagher et al., 2002, Am. J. Em. Med. v. 20; i4:287-290. Trained healthcare professionals administer the drug by injection and within 30 seconds of each injection, subjects rate their level of pain upon injection using a 100 mm visual analogue scale (VAS). A difference of 13 to 16 mm according to the VAS is considered clinically significant. Using this technique, it was demonstrated that the placebo formulation without phosphate caused significantly less pain than the placebo formulation containing phosphate at pH 6.3 and the current commercial formulation containing both etanercept and phosphate at pH 6.3.

The preparation and purification of etanercept for use in the pharmaceutical compositions and methods of the present invention can be accomplished by any standard method. Typically, etanercept is recombinantly expressed in CHO cells and secreted into the medium. The medium is collected, filtered and purified using, for example, various chromatographic techniques. For example, Protein A can be used to purify Fc domain containing polypeptides such as etanercept and is preferred as the first processing step. Other methods for purifying polypeptides are ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, SEPHAROSET™ heparin chromatography, anion exchange or cation exchange resin chromatography (such as a polyaspartic acid column), hydroxyapatite chromatography, gel - electrophoresis, dialysis and affinity chromatography and any combination of purification techniques known or not yet developed. Examples of applicable preparation and purification procedures can be found in US Pat. 8163522 (Brockhaus et al.) and 7648702 (Gombotz et al.).

Methods of the present invention.

The present invention also provides a method of formulating an etanercept pharmaceutical composition while debuffering and maintaining a pH within a target range, comprising formulating etanercept in a buffered formulation within that target range and exchanging the buffered formulation against an unbuffered formulation that is within or slightly below that target range, and harvesting the resulting etanercept pharmaceutical formulation. In a preferred embodiment, illustrated by way of example below, the method comprises formulating an etanercept pharmaceutical composition while debuffering and maintaining a pH in the range of 6.0 to 6.6, comprising formulating the etanercept-based pharmaceutical in a buffered formulation at a pH of 6 0 to 6.6, swapping the buffered formulation against an unbuffered formulation with a pH of 5.6 to 6.5, and harvesting the resulting pharmaceutical formulation. To provide an unbuffered etanercept formulation that maintains pH at

- 7 041785 range from 6.1 to 6.5, it is important to ensure an accurate determination of the pH value of both the original buffered formulation based on etanercept and the unbuffered formulation. For example, if the pH of the original etanercept buffer formulation is 7.2, then it must be adjusted with a strong acid such as HCl to a range of 6.1 to 6.5. Similarly, the unbuffered formulation used as a replacement must be titrated to a pH in the range of 5.6 to 6.5. Because the unbuffered replacement formulation does not contain a buffer agent, care must be taken when titrating.

In order to exchange a buffered formulation versus a non-buffered formulation, one skilled in the art can apply various buffer exchange techniques that are well known in the art. Dialysis uses selective diffusion across a semi-permeable membrane to remove unwanted small molecules from larger proteins. In one embodiment, the equilibrations are carried out sequentially until the required fold reduction in the concentration of the unwanted molecule is reached. For example, three consecutive equilibrations, each at 100-fold dilution or greater, can be used to achieve a concentration reduction of 1,000,000-fold or greater. Ultrafiltration and diafiltration are similar to dialysis in that they use a semi-permeable membrane. But unlike passive diffusion in dialysis, ultrafiltration and diafiltration involve the supply of solutions through a membrane under pressure using different techniques. As a rule, pressure and centrifugation are used. Another way to change the buffer can be done using gel filtration or size exclusion chromatography. There are many other chromatographic techniques that can also be used to perform buffer exchange that are well known to those skilled in the art, such as ion exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography.

After the buffered formulation is replaced with a non-buffered formulation, the methods of the present invention include collecting the resulting pharmaceutical formulation. At this stage, essentially all the buffer has been removed, but the pH is still maintained at the required levels. In the case of a pharmaceutical composition containing etanercept, the pH is maintained in the range of 6.0 to 6.6.

If necessary, the pharmaceutical composition can be further processed. For example, a surfactant may be added. In another example, if particles are to be removed, then the pharmaceutical composition can be filtered. Alternatively or additionally, the methods of the present invention also include the division of pharmaceutical compositions into aliquots in the form of a finished dosage form. Such finished dosage forms are distributed for end use by patients or health care providers. The pharmaceutical compositions of this invention are particularly suitable for parenteral administration, i. subcutaneously, intramuscularly, intravenously, intraperitoneally, intracerebrospinally, intraarticularly, intrasynovially and/or intrathecally. Parenteral administration can be by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers. Pharmaceutical compositions, if desired, may be presented in a vial, package or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispensing device may comprise a single dose syringe of a liquid formulation ready for injection. In another embodiment, the pharmaceutical composition is aliquoted into a cassette component for use with a reusable pen. In yet another aspect of the present invention, the pharmaceutical compositions may be provided packaged in or with a wearable injection device. In yet another embodiment, the pharmaceutical compositions may be aliquoted into a dosage form suitable for a needleless injector.

The pharmaceutical composition can also be divided into aliquots in a format suitable as a depot preparation. Such long acting formulations may be administered by implantation (eg subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, formulations can be modified with suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil) or ion exchange resins, or as sparingly soluble derivatives, eg, as a sparingly soluble salt.

In another embodiment, the present invention is directed to a kit or container that contains a pharmaceutical composition of the present invention. Instructions for the storage and use of pharmaceutical compositions may also be supplied with the kit. The container may be, for example, a disposable container, ie. container that contains a single dose of the composition of the present invention. It is understood that the disposable container may contain a single dose and a sufficient additional amount to ensure that the patient can receive a full single dose from the container, but not so much up to

- 8 041785 an additional amount so that this container can be used to administer a second dose. Examples of containers suitable for use in certain aspects of the present invention (whether disposable or refillable containers) include vials, syringes, and pens. Examples of suitable syringe pens include those described in US Pat. 777259 and PCT publications WO 2014/0089393, WO 2016/033496 and WO 2016/033507, each of which is incorporated herein by reference in its entirety.

The etanercept compositions and formulations of the present invention, as well as the syringes, pens, kits, and the like described herein, can be used in the treatment of patients with conditions that respond to etanercept treatment. Examples of such conditions include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and psoriasis. Methods for treating patients with etanercept are described, for example, in US Pat. Nos. 7,915,225; 8,119,605; 8,410,060;

The present invention will be more fully understood with reference to the following examples. However, the examples should not be construed as limiting the scope of the present invention.

Examples

Example 1 Stability testing of various formulations.

This example demonstrates the effect of pH and buffer on etanercept at 50 mg/mL, assessing the stability of a highly concentrated (100 mg/mL) solution without the addition of phosphate buffer. The following formulations were tested.

Table 1

pH screening formulations

Composition name Buffer Excipients Added polysorbate Final pH Conc. protein (mg/ml) A45SuT 10 mM acetate sodium 9% sucrose 0.004% PS20 4.5 50 A52SuT 10 mM acetate sodium 9% sucrose 0.004% PS20 5.2 50 A58SuT 10 mM acetate sodium 9% sucrose 0.004% PS20 5.8 50 50_SAST_100NaCl absent 25 mM L-arginine, 100 mM NaCl, 1% sucrose 0.004% PS20 6, 3 50 100_SAST_100NaCl absent 25 mM L-arginine, 100 mM NaCl, 1% sucrose 0.004% PS20 6, 3 100 PASST+BeOH 25 mM phosphate 25 mM L-arginine, 100 mM NaCl, 1% sucrose 0.9% benzyl alcohol 0.004% PS20 6, 3 50 PASST (control) 25 mM phosphate 25 mM L-arginine, 100 mM NaCl, 1% sucrose 0.004% PS20 6, 3 50

Materials: Enbrel drug in PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) at 50 mg/mL was used in this study. In the case of formulation with acetate and without buffer, the material was dialyzed to obtain new formulations (without polysorbate) and concentrated to 50 mg/mL using Centriprep MWCO 10000 membrane filter centrifuge tubes. Sample 50_SAS_100NaCl was also concentrated to 100 mg/mL (100_SAS_100NaCl) . Benzyl alcohol was introduced in the exact amount in the modern composition until

- 9 041785 final concentration of 0.9%. A 1% stock solution of Polysorbate 20 was prepared fresh and added in exact amounts to all formulations to a final concentration of 0.004%. All formulations were manually filled into 1 ml BD glass syringes to a volume of 0.5 ml and then capped using an ASPU vacuum stopper.

Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler InLab MicroProbe. Before measurements, the samples were heated to room temperature. Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 µl of sample and 290 mOsm osmolality standards were tested to ensure proper system performance. Exclusive HPLC was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software. Size exclusion HPLC with denaturation was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software.

Results: The pH of all formulations was maintained for 24 weeks.

table 2

pH value at specified times and at specified temperatures

Sample

A45SuT

A52SuT

A58SuT

50_SAST_100NaCl

100 SAST lOONaCl

PASST+BeOH

PASS control

pH value t=0 t=24 weeks 4°C 4°C 25°C 40°C 4, 60 4.66 4.66 4.61 5, 10 5, 13 5, 16 5, 08 5, 61 5.60 5.66 5.62 6, 24 6, 21 6, 22 6, 17 6, 32 6, 21 6, 20 6, 19 6, 27 6, 22 6, 22 6, 21 6.26 6.21 6.21 6.19

Table 3

Aggregation levels (peak B) obtained by SEC, % of total, at 4°C

Sample t=0 t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 0.9 eleven 1.1 1.2 1.2 A52SuT 1.2 1.4 1.5 16 1.4 A58SUT 1.0 1.2 1.2 1.3 1.3 50 SAST lOONaCl 1.1 1.3 1.3 1.3 1.4 100 SAST lOONaCl 1.1 1.4 1.5 1.5 16 PASST+BeOH 1.0 1.2 1.2 1.3 1.3 PASST control 1.0 1.1 1.2 1.2 1.3

Table 4

Aggregation levels (peak B) obtained by SEC, % of total, at 25°C

Sample t=0 t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 0.9 2.0 2.7 3.2 4.1 A52SuT 1.2 2.7 3, 1 3, 6 4, 6 A58SuT 1.0 1.9 2, 6 3, 1 4.4 50 SAST lOONaCl 1.1 1.9 2.4 2.8 3.7 100 SAST lOONaCl 1.1 2, 6 3.3 3, 9 5, 3 PASST+BeOH 1.0 1.9 2.4 2.8 3, 8 PASST control 1.0 1.8 2.2 2, 6 3.5

Table 5

Aggregation levels (peak B) obtained using SEC, __________% of the total, at 40 ° C _________________

Sample t=0 t=2 weeks t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 0.9 5, 6 9.0 11.9 11.9 9, 6 A52SuT 1.2 6, 3 12.9 13, 6 16, 4 17.9 A58SuT 10 5, 1 9, 1 14.7 18.4 24.5 50 SAST lOONaCl 1.1 3, 9 6, 8 12.1 16.0 26.0 100 SAST lOONaCl 1.1 5, 9 10, 8 18.5 24.0 37.8 PASST+BeOH 1.0 6, 3 12.0 21.4 28.1 45.5 PASST control 1.0 4.0 6, 9 12.1 16, 1 26, 7

- 10 041785

Table 6

Low molecular weight compounds (truncated according to dSEC analysis) at 4°C

Sample t=0 t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 16 1.3 1.4 2.3 2.5 A52SuT 1.2 16 1.3 1.7 1.7 A58SuT 1.4 1.7 1.5 1.4 1.4 50 SAST lOONaCl 0.9 1.5 1.4 1.4 1.5 100 SAST lOONaCl 10 1.5 1.7 2.0 2, 6 PASST+BeOH 1.0 1.5 1.2 1.4 1.3 PASST control 1.2 1.4 1.4 1.7 16

Table 7

Low molecular weight compounds (truncated according to dSEC analysis) at 25 °C

Sample t=0 t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 16 3, 6 5, 9 8.4 13.1 A52SuT 1.2 2.9 2.9 5, 8 9, 2 A58SuT 1.4 2.2 2.8 4.0 5, 7 50 SAST lOONaCl 0.9 2.2 2.4 2.9 4.7 100 SAST lOONaCl 10 2.9 3.2 4.4 6, 9 PASST+BeOH 10 2.2 2.2 3.3 4.4 PASST control 1.2 16 2.1 3.2 4, 8

Table 8

Low molecular weight compounds (truncated according to dSEC analysis) at 40°C

Sample t=0 t=2 weeks t=4 weeks t=8 weeks t=12 weeks t=24 weeks A45SuT 16 9, 7 16, 7 28.3 38.1 55.0 A52SuT 1.2 6.0 11.3 18.6 26.0 39.6 A58SuT 1.4 4.0 7.7 12.2 18.2 27.5 50 SAST lOONaCl 0.9 3, 6 5, 9 8, 6 12.8 18.7 100_SAST_l0ONaC 1 1.0 3.5 5, 7 9, 6 12.9 19, 4 PASST+BeOH 1.0 3, 6 5, 8 9, 8 13.2 20.5 PASST control 1.2 3.3 5, 9 100 12.8 20.2

Conclusion: During extended storage at 25 and 40°C, the lower pH formulations A45SuT, A52SuT and A58SuT - exhibited undesirable levels of degraded small molecule compounds or truncated compounds when analyzed using denaturation size exclusion chromatography. The highly concentrated 100_SAST_100NaCl formulation stored at 25 and 40°C began to show an increase in high molecular weight aggregates when analyzed using size exclusion chromatography, but at 4°C behaved similarly to the current commercial formulation. PASST+BeOH (which is a modern commercial formulation modified by the addition of 0.004% polysorbate 20 and 0.9% benzyl alcohol) behaved similarly to the modern commercial formulation at both 4°C and 25°C, but exhibited an increase in high molecular weight compounds according to SE-HPLC at later times during storage at an elevated temperature of 40°C. However, the 50_SAST_100NaCl formulation maintained high and low molecular weight compound levels that were comparable to the current commercial formulation at all temperatures, even in the absence of phosphate buffer.

Example 2. Study of pain sensations.

This study was a single center, randomized, single blind, crossover design in which 48 healthy men and women received a single SC injection of 6 solutions.

The test formulations (details below in Table 9) were administered by a trained medical professional in 6 individual sequences to 8 subjects randomized to each sequence. Injections were given into each quadrant of the anterior abdominal wall approximately 1 hour apart. Within 30 seconds after each injection, subjects assessed their level of pain associated with the injection using a 100 mm visual analogue scale (VAS). Adverse events were collected from the start of the first injection up to 30 days after the first injection. Phone calls for safety follow-up were made on day 2 (24 hours after the sixth injection) and day 31 (±2 days).

- 11 041785

Test formulations

Table 9

Solution Description Volume Composition A. Negative pain control 10 10 mM sodium acetate, 9% (w/v) sucrose, 0.004% (w/v) polysorbate 20, pH 5.2 IN. Commercial placebo formulation 0.098 25 mM sodium phosphate, 25 mM L-arginine, 100 mM sodium chloride, 1.0% (w/v) sucrose, pH 6.3 WITH. Commercial placebo formulation with benzyl alcohol 1.0 100 mM sodium chloride, 25 mM sodium phosphate, 25 mM L-arginine, 1.0% (w/v) sucrose, 0.01% (w/v) polysorbate 20, 0.9% (w/v) benzyl alcohol D. The tested composition without sodium phosphate 1.0 100 mM sodium chloride, 25 mM L-arginine, 1.0% (w/v) sucrose, 0.01% (w/v) polysorbate 20 E. Tested composition without 0.51 100 mM sodium chloride, 25 mM L-arginine, 1.0% (w/v) sodium phosphate sucrose, 0.01% (w/v) polysorbate 20 F. Commercial composition on based on etanercept, 50 mg/ml 0.98 50 mg/ml etanercept in solution consisting of 100 mM sodium chloride, 25 mM sodium phosphate, 25 mM L-arginine, 1% sucrose, pH 6.3

Statistical Methods: All analyzes were performed with a safety analysis population that consisted of all subjects who received at least one solution. To provide 93.4% power to detect a difference of 15 mm between solutions (α=0.05, two-tailed), a sample size of 48 subjects (8 per sequence) was chosen. A difference of 13 to 16 mm on the VAS is considered clinically significant (Gallagher et al., 2002, Am. J. Em. Med. V. 20; i4:287-290).

Summary statistics (mean, standard deviation [SD], standard error [SE], median, minimum, maximum) were calculated for VAS scores per solution. VAS scores were analyzed using an analysis of variance (ANOVA) model that included sequence, solution, and period as independent variables and subject in sequence as a random effect. No adjustments were made for multiple comparisons.

Received indicators of the average difference between VAS scores for primary and secondary comparisons, the corresponding 95% confidence intervals (95% CI) and p-values.

Table 10

Brief description of VAS scores

Directly but after injections Solution r A Solution B Solution p C Solution p D Solution E Solution op F N 48 48 48 48 48 48 Average 19.6 53, 6 28.7 29, 8 29, 6 53.4 SD 18.0 27.9 23.5 26, 4 24.3 32.4 SE 2, 6 4.0 3.4 3, 8 3.5 4.7 Median 13.0 59.0 24.0 21.5 21.0 49.0

- 12 041785

Minimum, maximum 0.66 1.98 1.99 0.94 1.86 2, 100

Conclusion: Both Solution C (representing placebo with benzyl alcohol, non-product specific) and Solution D (representing placebo without sodium phosphate, non-product specific), mean VAS scores were significantly lower than solution B (etanercept placebo; p<0.001), indicating relatively less pain at the injection site with the two solutions. No significant differences were found in mean VAS scores between solutions C and D, between solution B (etanercept placebo) and solution F (etanercept formulation), or between different injection volumes (0.51 and 1.0 ml). Solution A (negative control in the pain study) was associated with the least pain compared to all other solutions. Seven subjects had one or more adverse events. All adverse events were CTCAE Grade 1, ie. were injection site reactions that were not serious.

Example 3 Long-term stability testing of candidate formulations.

A long-term study was performed to monitor the stability of etanercept in several new candidate formulations at 50 mg/mL. Stability was assessed by filling 1 ml in 1 ml syringes with non-removable glass needle using SE-HPLC, HIC HPLC, dSEC HPLC and suspended solids assessment (HIAC) after storage at 4, 25 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 confirm no pH shift. The results of the study showed that the tested formulations remained similar to the current commercial formulation after 12 weeks of storage at an elevated temperature of 40°C, and after 24 weeks of storage at the recommended storage temperature of 2-8°C and at an elevated temperature of 25°C.

Table 11

Formulation parameters at 50 mg/ml etanercept

Composition name Buffer Other excipients pH value PAS ST (control) 25 mM phosphate 25 mM L-arginine, 100 mM NaCl, 1% sucrose, 0.010% polysorbate 20 6, 3 SAST_100NaC 1 Absent absent 25 mM L-arginine, 100 mM NaCl, 1% sucrose, 0.010% polysorbate 20 6, 3 SAST_120NaC 1 25 mM L-arginine, 120 mM NaCl, 1% sucrose, 0.010% 6, 3 polysorbate 20

Materials: Enbrel drug in PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) at 50 mg/mL was used in this study. The material was diafiltered in PASS and SAS_100NaCl (25 mM L-arginine, 100 mM NaCl, 1% sucrose) at 50 mg/ml and then ultrafiltered to ~75 mg/ml. Formulations with a concentration of 50 mg/ml were prepared by diluting the material PASS and SAS after UF/DF with the appropriate solution. SAST_120NaCl was prepared by diluting 75 mg/mL SAS material using a concentrated NaCl stock solution to a final concentration of 120 mM NaCl. A 1% stock solution of Polysorbate 20 was prepared fresh and added to all formulations to a final concentration of 0.010%. All formulations were manually filled into 1 ml BD glass syringes to a volume of 1 ml and then capped using an ASPU vacuum stopper.

Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler InLab MicroProbe. Before measurements, the samples were heated to room temperature. Protein concentration measurements based on absorbance at 280 nm for all samples were performed at room temperature using the DropSense96 UV/Vis Lab Chip DS system. Each sample was measured neat in at least triplicate (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 290 mOsm osmolality standards were tested to ensure proper system performance. Exclusive HPLC was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software. Hydrophobic interaction HPLC was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software with absorbance at 215 nm. Size exclusion HPLC with denaturation was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software. The analysis of particles invisible to the naked eye was carried out using the system

- 13 041785 HACH HIAC/Royco particle counter equipped with HRLD-150 laser and Pharm Spec software. All samples were diluted with PASS formulation buffer to 25 mg/mL. Samples were thoroughly mixed, uncapped, and degassed for 2 hours at 75 torr prior to analysis. Four samples of 1.0 ml each (without tare volume) were analyzed, with the first sample discarded and averaging of the remaining three samples. Data for particle sizes of 2, 5, 10 and 25 µm were collected at all time points. Results take into account dilution and are presented as cumulative occurrence per ml.

Results and Discussion: The pH of all formulations was measured at time zero and after 12 weeks of storage at all temperatures. No trends were found to change depending on storage time or temperature. The measured pH values for all samples can be found in Table. 12. No pH shift detected after 52 weeks at 4°C, after 24 weeks at 25°C, or after 12 weeks at 40°C; all samples met the acceptance criteria of ±0.2 pH units from the target pH of 6.3.

Table 12

Measured pH value for samples

Composition abbreviation t=0 t=52 weeks 4°C t=24 weeks 25°C t=12 weeks 40°C PASS 6, 27 6, 30 6, 32 6.28 SAST_100NaCl 6, 23 6, 15 6, 25 6.20 SAST_120NaCl 6, 22 6.16 6, 24 6, 15

Protein concentration in all formulations was tested at time zero. The results of protein concentration for all samples can be presented in table. 13. All samples met the acceptance criteria.

Table 13

Protein concentration indicators

Osmolality was tested only at time zero. Osmolality results for all samples can be found in Table 1. 14. All formulations had their target osmolality. Due to differences in buffer and excipient levels, osmolality was not expected to be the same for different formulations.

Table 14

Osmolality indicators

SE-HPLC was performed to control the levels of aggregation depending on the parameters of the composition, time and temperature. Peak B represents the amount of high molecular weight compounds (aggregates) formed. The results showed no difference in peak B between the PASST control and formulations without buffer at 4 and 25° C., with slight differences observed after 12 weeks of storage at 40° C. (FIG. 1). Peak B represents the total number of aggregates detected by SE-HPLC for these formulations. All samples remained usable (peak B <6%) after 52 weeks of storage at 4°C, after 24 weeks of storage at 25°C and after 12 weeks of storage at 40°C.

Denatured SE-HPLC was used to monitor for truncated LMW compounds. The results showed similar trends in particle HMW, main peak, LMW between formulations at 52 weeks (FIG. 2).

Changes in mispacked aggregates were monitored using HIC HPLC. The results at all temperatures tested showed no difference in peak 3 between the PASST control and the formulations without buffer (FIG. 3). All samples remained within acceptable ranges (peak 1 <5%, peak 2 >70%, peak 3 <28%) after 52 weeks of storage at 4°C, after 24 weeks of storage at 25°C, and after 12 weeks of storage at 40°C WITH.

Particles invisible to the naked eye were monitored by chiaroscuro particle counting (HIAC). The results were consistent with accumulated PFS data and were the same for all formulations at all temperatures at 12 weeks. No trends can be established based on this dataset as one vial of three pooled syringes was used at each time point and there is a high level of inter-syringe variability

- 14 041785 in terms of the size of the contribution of drops of silicone oil.

Conclusion: The long-term stability of several new modified candidate formulations and current commercial formulation with the addition of polysorbate was evaluated at 4, 25 and 40°C. Using SE, dSEC or HIC HPLC analyzes, as well as particle counting in chiaroscuro mode, there were no significant differences between formulations after storage for 52 weeks at 4°C and 24 weeks at 25°C; while using HPLC analyzes revealed minor differences after 12 weeks of storage at 40°C. No pH shift was detected and all formulations remained within acceptable ranges. The results of the study showed that the SAST_120NaCl and SAST_100NaCl formulations at 50 mg/mL were stable and similar to the current commercial formulation after 12 weeks at the recommended storage temperature of 2 to 8°C.

Example 4 Freeze/Thaw and Long Term Stability of Best Modified Candidate Formulations in Stainless Steel Containers.

A freeze/thaw cycle study was conducted to monitor the stability of etanercept in three new candidate formulations at a concentration of 50 mg/mL. The formulations compared to the current commercial PASS formulation (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) were 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). Stability against aggregation at temperature cycling from -30 to 4° C. in 55 ml stainless steel cryovessels was assessed using SE-HPLC for up to five freeze/thaw cycles inclusive.

In addition, a long-term study was conducted to monitor the stability of etanercept in the new candidate formulation at a concentration of 50 mg/ml. The formulation compared to the current commercial PASS formulation (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). Storage stability in 10 and 55 ml stainless steel cryo-vessels was assessed using SE-HPLC, HIC HPLC, dSEC HPLC and suspended solids assessment (HIAC). Storage temperatures and times were -30°C for up to 36 months and 4°C for up to 12 months. Results for 52 weeks are presented in this document.

Results: The pH values for all formulations remained constant at time point 52 weeks and through five freeze/thaw cycles.

Table 15

pH value, protein concentration and osmolality

Sample Conc. (mg/ml) Osmolality (mOsm) pH value t=0 52 weeks, 30°C 52 weeks, 4°C PASS 49.5 304 6, 34 6.20 6, 22 SAST_120NaCl 51.4 303 6, 27 6, 19 6.20

Table 16

pH, protein concentration and osmolality during freeze/thaw cycles

Sample Conc. (mg/ml) Osmolality (mOsm) pH value 0 freeze/thaw 3 freeze/thaw 5 freeze/thaw PASS 47.9 310 6, 30 6.29 6, 27 SAST_100NaCl 48.8 259 6, 19 6, 18 6.16 SAST_120NaCl 48.1 294 6, 17 6, 19 6, 18 SAS_120NaCl 48.6 300 6, 17 6, 17 6, 18

HIAC did not show any trend towards >10 µm particles, although SAST_120NaCl did show a slight increase in >2 and >5 µm particles. As shown in FIG. 4-6, analysis by HIC, dSEC, or SEC showed no significant differences between formulations. SEC did not reveal significant changes between formulations after exposure to five freeze-thaw cycles. See fig. 7.

Conclusion: The study results to date have shown that the new formulation tested remained similar to the current commercial formulation after 52 weeks of storage in stainless steel cryo-vessels at -30°C as well as at 4°C.

- 15 041785

Example 5 Change to SAS and PASS solutions.

The purpose of these examples was to dialyze various etanercept formulations in TMS (Tris, mannitol, sucrose) to a test formulation (L-arginine, sucrose, NaCl) and compare the final pH to the target pH.

Materials: etanercept: 25 mg/ml in TMS (10 mM Tris-HCl, 4% mannitol, 1% sucrose, pH 7.4); SAS_100NaCl solution (100 mM NaCl, 25 mM L-arginine HCl, 1% sucrose, pH 6.3) for dialysis; PASS buffer (25 mM phosphate, 100 mM NaCl, 25 mM L-arginine HCl, 1% sucrose, pH 6.3); centrifuge tubes with membrane filter Centriprep MWCO 10000; cassettes for dialysis with a volume of 3-12 ml Slide-A-Lyzer, MWCO 10000; pH meter Mettler Toledo MP220 and Mettler Toledo InLab MicroProbe.

Methods: For the UF/DF example, 25 mg/mL etanercept in TMS was concentrated to ~50 mg/mL by ultrafiltration using MWCO 30K Pellicon 3 cassettes on a Millipore Pellicon-2 mini-system. The material was then diafiltered against a solution of SAS_100NaCl or PASS for 7 diafiltration volumes and then concentrated by ultrafiltration to 100 mg/mL. As a dialysis example, 25 mg/mL etanercept in TMS was concentrated to 50 mg/mL using centripreps MWCO 10000 membrane filter centrifuge tubes. -meter Mettler Toledo MP220 and InLab MicroProbe. The material was then dialyzed using MWCO 10000 Slide-A-Lyzer dialysis cassettes. 9.5 ml 50 mg/ml etanercept in TMS was added to the cassette and exchanged against 1000 ml SAS100. Three replacements were carried out to achieve a 1,000,000-fold replacement. The first change was made at 5 pm on day 1 and continued throughout the night. The second 1000 ml change was done at 8:30 am on day 2. The third and last change was done at 12:30 pm on day 2. At 5:00 pm on day 2, the protein was removed from the dialysis cassette (11 ml removed) and the pH was measured using the same Mettler Toledo MP220 pH meter. The measured pH was 6.98.

Results: a summary of the results is shown in Table. 17.

Table 17

pH value when using various replacement methods and solutions

Replacement method Sample name Solution pH for pH value before the method pH after method Quantity substitutions filtration substitutions substitutions UV/DF PASS, pH 6.3, 100 mg/mL 6, 34 7.56 6, 34 7 volumes UV/DF SAS_100NaCl indicator pH 6.3, 100 mg/ml 6, 38 7.56 6.98 7 volumes Dialysis SAS_100NaCl indicator pH 6.3, 50 mg/ml 6.29 7.56 6.98 1000000 times replacement

Conclusion: When samples were ultrafiltered/diafiltered from solution prior to replacement at pH 7.56 with PASS buffer, the target pH of 6.34 was achieved. However, when the samples were ultrafiltered/diafiltered into a solution of SAS_100NaCl, then the achieved pH of the material after dialysis was 6.98, which was higher than expected and was not close to the final target pH of 6.3. Using dialysis as a replacement method for SAS_100NaCl led to the same results.

Example 6: UF/DF pool.

Introduction: The composition solution chosen after this study was called SAS (120 mM sodium chloride, 25 mM L-arginine, 1% sucrose, pH 6.3) without the addition of phosphate buffer. Since the previous example demonstrated that it was difficult to reach the target pH of 6.3 with both dialysis and UF/DF when starting with etanercept in the sample at pH 7.56, a different substitution method for the SAS formulation was needed. Two methods were evaluated using different final UF/DF starting material: 1) column 3 intermediate pool (AEX) as starting material and 2) Enbrel drug in PASS formulation buffer (PASS DS intermediate pool) as starting material. Each method is described below and summarizes the development of the final process step for a UF/DF plant to produce 50 g/L etanercept in SAS formulation, including preparation of the SAS formulation solution, conditioning, and processing of the UF/DF final feed.

Methods: The SAS formulation solution consists 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 10N sodium hydroxide. NaOH. The volume of titrant required to achieve a certain pH range was 4.4 µl/l of the SAS composition solution. During the final process step with the UF/DF SAS unit, after the membranes were equilibrated at 10 L/m ² with the SAS solution, the pH of the permeate remained close to the pH of the WFI, but not the pH of the SAS solution. Without wishing to be bound by a particular theory, this is believed to be the result of the low buffer capacity of the SAS formulation solution. The expected permeate conductivity range after membrane equilibration using the solution preparation range for the SAS formulation is 12-16 mS/cm. A higher pH after equilibration is expected than the SAS solution, but this should not be a cause for concern or indicate that the membranes are not balanced.

AEX Intermediate Pool Feed: Prior to transferring the AEX Intermediate Pool to the UF/DF tank retentate tank, the pool was conditioned with 2 M HCl to a target pH of 6.3 (acceptable range 6.2-6.4). The volume of titrant required to achieve a certain pH range was about 2.8 ml/l of the AEX intermediate pool.

Eight examples performed during the development of the final technological operation of the plant for UF / DF SAS using the intermediate pool AEX as the source material are shown in table. 18. Two parameters were investigated: the pH of the conditioned AEX intermediate pool and the pH of the SAS formulation solution. The first three runs analyzed pH, conductivity, osmolality, protein concentration and product quality. In runs 4 through 7, only pH, conductivity, osmolality, and protein concentration were measured to determine the effect of formulation solution pH and loading pH on UF/DF pool pH.

Table 18

AEX Intermediate Pool Source Material:

pH values of the load, replacement solution and final UF/DF pool

Run number Load pH target Target pH SAS Pool pH UF/DF 1 6, 3 6, 3 6.26 2 6, 3 6, 3 6, 33 3 6, 3 6, 3 6, 22 4 6, 3 5, 6 6, 22 5 6, 2 5, 3 6, 06 6 6, 4 7.3 6, 94 7 6, 2 5, 6 6, 14 8 6, 4 6, 5 6, 43

Results: The product quality results for the final pool of UF/DF SAS obtained using the intermediate pool AEX as starting material are shown in Table 1. 19. The stage exit for Run 1 was outside the acceptance criteria; however, this was most likely a laboratory processing artifact and was not considered significant for the study results. All three UF/DF SAS final runs also met product quality acceptance criteria using SEC and HIC analysis as described above.

Table 19

AEX Intermediate Pool Feedstock: UF/DF SAS Final Pool Product Quality

Parameter Acceptance Criteria End pool UF/DF SAS Run 1 Run 2 Run 3 pH value 6.1-6.5 6.26 6, 22 6, 33

- 17 041785

Protein concentration 49-51 50, 08 49, 68 49, 90 Yield per stage (%) 95-103 93.4 99.5 100.5

Stability of the conditioned intermediate pool AEX.

The AEX conditioned intermediate pool can be stored for up to 52.6 hours at controlled room temperature (CRT). The pH of the pool during storage is shown in FIG. 8.

UF/DF pool stability.

The final UF/DF SAS pool created using the AEX staging pool as starting material can be stored for up to 96.3 hours at CRT. The pH and conductivity values during storage are shown in FIG. 9A and B. During 96.3 hours of storage, the pH and conductivity remain within acceptable limits.

PASS DS Intermediate Pool Feedstock: Conditioning before transferring the PASS DS Intermediate Pool to the UF/DF Retentate Tank is not required as the PASS DS Intermediate Pool is already in an acceptable pH range. Also, since the starting material is Enbrel DS at 50 mg/mL formulated in PASS, the pool does not need to be concentrated to 50 g/L as it is already at the correct concentration for diafiltration.

One example, carried out during the development of the final technological operation in the installation for UF/DF SAS in order to evaluate the source of the starting material, is given in table. 20. In this example, the PASS DS intermediate pool was used as starting material and analyzed for pH, conductivity, osmolality, protein concentration, and product quality.

Table 20

PASS DS Intermediate Pool Feed: Load pH, Exchange Solution, and UF/DF Final Pool

Run number Target pH Target pH Pool pH UF/DF while loading SAS solution 1 6, 3 6, 3 6, 23

Results: Product quality results for the final pool of UF/DF SAS obtained using the intermediate pool PASS DS as starting material are shown in Table 1. 21. The stage exit for Run 1 was outside the acceptance criteria; however, this was most likely a laboratory processing artifact and was not considered significant for the study results. The final pool of UF/DF SAS also met product quality acceptance criteria using SEC and HIC analysis as described above.

Table 21

PASS DS Intermediate Pool Feedstock: UF/DF SAS End Pool Product Quality

Parameter Acceptance Criteria End pool UF/DF SAS Run 1 pH value 6.1-6.5 6, 23 Protein concentration (mg/ml) 49-51 49, 60 Yield per stage (%) 95-103 105.7

PASS DS staging pool stability.

The PASS pool does not require conditioning prior to UF/DF treatment with SAS solution as this intermediate pool already has a target pH of 6.3. A pool storage study was not performed for this staging pool as the pool conditions did not change relative to Enbrel PASS DS. The pool can be stored up to 96 hours at 25°C.

UF/DF pool stability.

The final UF/DF SAS pool created using the PASS DS staging pool as starting material can be stored for up to 96.3 hours at CRT. The pH and conductivity values during storage are shown in FIG. 9A and B. During 96.3 hours of storage, the pH and conductivity remain within acceptable limits.

The stability of the SAS composition solution.

The SAS formulation solution can be stored at CRT for up to 28 days. The pH and conductivity values are shown in FIG. 10A and B. During 42 days of storage in small chambers to study the stability of

- 18 041785 stainless steel with little headspace shows that the solution of the SAS composition maintains a pH in the range of 5.6 to 6.5. At time points day 35 and day 42, precipitation was observed. The score at day 21 of 5.09 appears to be an outlier because subsequent time points meet the proposed acceptance criteria.

Conclusion: The final process step in the UF/DF plant allows a product with a SAS composition at 50 g/l and a consistent product quality compared to a modern commercial product with a PASS composition according to the following method recommendations: 1) using either an intermediate pool of AEX, or intermediate pool PASS DS as source material; 2) the SAS solution can be stored for at least 28 days at CRT and maintain a pH in the range of 5.6 to 6.5; 3) the conditioned intermediate pool AEX can be stored at CRT for at least 52.6 hours and maintain a pH of 6.3 ± 0.1; and 4) the pool of UF/DF in the SAS can be stored at CRT for at least 96.3 hours and maintain a pH in the range of 6.1 to 6.5 and a conductivity in the range of 10 to 14 mS/cm.

Example 7 Alternative Isotonic Formulations

The purpose of this example was to determine the effect of elevated levels of arginine, sucrose or sodium chloride on the aggregation and stability of etanercept at a concentration of 75 mg/ml at 40°C. The levels of each of these excipients were increased to maintain the isotonicity of the formulation without the addition of phosphate buffer. In addition, histidine was evaluated as a buffer for phosphate replacement. Data on the tested compositions are summarized in table. 22.

Table 22

Formulations for alternative isotonic formulations at pH 6.3 with 0.01% PS20

Name Buffer L-arginine (mM) NaCl (mM) sucrose composition (weight. %) PASS 25 mM phosphate 25 100 1% SAST lOONaCl absent 25 100 1% SAST 30Arg absent thirty 100 1% SAST 35Arg absent 35 100 1% SAST 40Arg absent 40 100 1% SAST 2Suc absent 25 100 2% SAST 120NaCl absent 25 120 1% HAS ST 10 mM histidine 25 100 1%

Materials: Enbrel drug in PASS (25 mM phosphate buffer, 25 mM L-arginine, 100 mM NaCl, 1% sucrose) at a concentration of 50 mg/mL was used in this study. The material was dialyzed into new formulations (no polysorbate) and concentrated to 75 mg/mL using Centriprep MWCO 30000 membrane filter centrifuge tubes. A 1% stock solution of polysorbate 20 was prepared fresh and added to all formulations to a final concentration of 0.01%. All formulations were manually filled into 1 ml BD glass syringes to a volume of 1.0 ml and then capped using an ASPU vacuum stopper.

Methods: pH was measured using a Mettler Toledo pH meter in combination with a Mettler MicroProbe. Before measurements, the samples were heated to room temperature. Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 µl of sample and 290 mOsm osmolality standards were tested to ensure proper system performance. Exclusive HPLC was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software.

Results: concentration, pH and osmolality are shown in table. 23. The degree of aggregation at a concentration of 75 mg/ml at 40°C was similar to the composition based on the commercial formulation for all formulations without the addition of phosphate with increased levels of L-arginine, sucrose and NaCl, as shown in table. 24. In addition, the use of histidine instead of phosphate as a buffer led to an increase in the degree of aggregation at 40°C.

- 19 041785

Table 23

Concentration, pH and osmolality for alternative isotonic formulations

Conc. Sample (mg/ml) pH value Osmolality PAS ST 76.3 6.27 308 SAST_100NaCl 76.8 6.30 266 SAST_30Arg 74.9 6.28 271 SAST_35Arg 76.9 6.27 280 SAST_40Arg 76.5 6.26 288 SAST_2Suc 75.2 6.26 291 SAST_120NaCl 77.1 6.24 301 HAS ST 76.3 6.36 272

Table 24

Aggregate/HMW levels according to SEC, % of total, at 40°C

Sample 0th a week 1st a week 2nd a week 4th a week 8th a week 12th a week PAS ST 2.3 5, 3 6, 6 12.0 16, 1 26, 1 SAST_100NaCl 2.3 50 6, 4 11.0 15, 3 24.1 SAST_30Arg 2.4 5, 1 6, 6 11.3 15, 9 24.8 SAST_35Arg 2.4 5, 2 6, 8 11.5 16, 7 25, 3 SAST_40Arg 2.4 5, 1 6, 7 11.0 16, 1 23.7 SAST_2Suc 2.4 4.9 6, 6 10.7 16, 2 23.4 SAST_120NaCl 2.4 50 6, 9 11.0 16, 7 23.7 HAS ST 2.3 6, 2 9, 1 14.8 22.9 32.3

Example 8 Stability of Formulations with Different Levels of Polysorbate 20

Long-term stability control studies were performed on etanercept at 0, 0.005, 0.01, and 0.015% polysorbate 20 in SAS at 50 mg/mL etanercept. In addition, a SAST formulation was tested with a high concentration of 100 mg/ml etanercept. Stability was assessed by filling 1 ml in 1 ml syringes with non-removable glass needle using SEHPLC, dSEC HPLC and suspended solids assessment (HIAC) after storage at 4, 25 and 40°C. Osmolality, pH and protein concentration were only tested at time zero. The results of the study showed that the 50 mg/mL formulations tested remained similar to the current commercial formulation after 24 weeks at the recommended storage at 2-8°C, as well as at elevated temperatures of 25 and 40°C. The 100 mg/mL SAST formulation performed similarly to the 50 mg/mL formulations in terms of pH and particles invisible to the naked eye; differences in aggregation levels according to SEC were attributed to protein concentration.

- 20 041785

Table 25

Formulation parameters at 50 mg/ml etanercept

Composition name Buffer Other excipients Polisor baht Conc. protein (mg/ml) PASS 25 mm phosphate 25 mM L-arginine, 100 mM NaCl, 1% sucrose 0 50 SAS00OT Absent 25 mM L-arginine, 120 mM NaCl, 1% sucrose 0 50 SAS005T Absent 25 mM L-arginine, 120 mM NaCl, 1% sucrose 0.005 50 SAS01 FROM Absent 25 mM L-arginine, 120 mM NaCl, 1% sucrose 0.01 50 SAS015T Absent 25 mM L-arginine, 120 mM NaCl, 1% sucrose 0.015 50 100_SAS0 UT Absent 25 mM L-arginine, 120 mM NaCl, 1% sucrose 0.010 100

Materials: Enbrel drug in TMS (10 mM Tris buffer, 4% mannitol, 1% sucrose) at 25 mg/mL was used in this study. The base volume used for the SAS formulation was titrated to pH 6.3. The material was ultrafiltered to ~50 mg/ml etanercept, then diafiltered into 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 a concentration of etanercept 50 mg/ml. The material for the high concentration variant was then ultrafiltered to 100 mg/ml etanercept. A 1% stock solution of Polysorbate 20 was prepared fresh and added to the formulations to the final concentration as set forth in Table 1. 25. Manually fill all formulations into 1 ml BD glass syringes to a volume of 1 ml and then stopper using an ASPU vacuum stopper.

Results and Discussion: The pH of all formulations was measured at time zero and after 12 weeks at 40°C and 24 weeks at 4 and 25°C. No changes were found depending on the time or temperature of storage. The measured pH values for all samples can be found in Table. 26. No pH shift was observed after 12 weeks at 40°C or 24 weeks at 4 and 25°C, and all samples met the acceptance criteria of ±0.2 pH units from the target pH of 6.3. Protein concentration and osmolality in all formulations were tested at time zero. Results for protein concentration and osmolality for all samples can be found in Table. 26.

Table 26

Concentration, osmolality and pH

Sample Conc. (mg/ml) Osmolality (mOsm) pH value, t=0 Show pH cal, 24 weeks, 4°С Show pH cal, 24 weeks, 25°С Show pH tel, 12 weeks, 40°C PASS 51.6 318 6.32 6, 34 6, 34 6.33 SAS00OT 52.1 306 6.33 6, 31 6, 33 6.37 SAS005T 51.2 304 6.33 6, 30 6, 30 6.32 SAS01OT 51.5 304 6.34 6, 30 6, 30 6.36 SAS015T 51.5 301 6.30 6, 30 6, 30 6.35 100_SAS010T 102.8 304 6.32 6, 28 6.29 6.36

SE-HPLC was performed to control the levels of aggregation depending on the parameters of the composition, time and temperature. Peak B represents the amount of high molecular weight compounds (aggregates) formed. The results showed no difference in peak B between the PASS control and no buffered formulations at all temperatures and respective protein concentrations (Tables 27-29). Peak B represents the total number of aggregates detected by SE-HPLC for these formulations. All 50 mg/mL samples remained viable (peak B <6%) after 24 weeks of storage at 4 and 25°C and after 2 weeks of storage at 40°C.

Particles invisible to the naked eye were monitored by chiaroscuro particle counting (HIAC). The results were consistent with accumulated PFS data and were the same for all formulations at all temperatures after 24 weeks (Table 30).

- 21 041785

Table 27

Peak B analysis by SEC, % of total, 4°C

Sample t=0 weeks t=4 weeks t=8 weeks t=12 weeks t=24 weeks PASS 1.7 1.7 1.7 1.7 18 SAS00OT 1.7 18 Ι,θ Ι,θ 1.9 SAS005T 1.7 1.8 18 1.8 1.9 SAS 01 FROM 1.7 Ι,θ Ι,θ 1.9 1.9 SAS015T 1.7 Ι,θ 18 Ι,θ 1.9 100_SAS010T 1.9 2.0 2.1 2.2 2.4

Table 28

Peak B analysis by SEC, % of total, 25°C

Sample t=0 weeks t=2 weeks t=4 weeks t=8 weeks t=12 weeks t=24 weeks PASS 1.7 2.1 2.4 2.9 3.5 4.8 SAS00OT 1.7 2.1 2.5 2.9 3.5 50 SAS005T 1.7 2.2 2.5 2.9 3.5 4.9 SAS01 FROM 1.7 2.1 2.5 thirty 3, 6 4.8 SAS015T 1.7 2.2 2.4 thirty 3.5 4.8 100 SAS01 FROM 1.9 2.8 3.4 4.4 5, 4 7, 6

Table 29

Peak B analysis by SEC, % of total, 40°C

Sample t=0 weeks t=2 weeks t=4 weeks t=8 weeks t=12 weeks PASS 1.7 5, 7 10.4 17.4 20.6 SAS00OT 1.7 4.9 θ, 9 15, 4 19, 2 SAS005T 1.7 5, 2 9, 1 15, 9 19, 4 SAS01 FROM 1.7 5, 2 9, 3 15, 8 19, 2 SAS015T 1.7 5, 4 9, 4 15, 9 19.0 100 SAS01 FROM 1.9 9.0 15, 6 24.5 27.1

Table 30

Particles/ml (chiaroscuro mode), 4°С

2 µm 10 µm 2 5 µm t=0 weeks t=24 weeks t=0 weeks t=24 weeks t=0 weeks t=24 weeks PASS 19197 3422 1785 670 25 67 SAS00OT 13004 4489 1265 762 60 104 SAS005T 12344 1292 502 59 7 4 SAS01 FROM 11964 2254 1058 167 9 2 SAS015T 11388 7283 934 591 2 4 100 SAS01 FROM 58087 8931 559 816 3 0

Conclusion: The long-term stability of the modified candidate formulation at 50 mg/ml etanercept concentration with polysorbate levels from 0 to 0.015% and the current commercial formulation was evaluated at 4, 25 and 40°C; in the case of SAS010T formulations, a variant with a high concentration of 100 mg/ml etanercept was also tested. No significant differences were observed between formulations at respective protein concentrations at 24 weeks using SE, dSEC, or HIC HPLC assays, as well as using chiaroscuro mode. No pH shift was detected and all formulations remained within acceptable ranges. The results of the study showed that the formulations of SAST_120NaCl at 50 mg/ml were stable and similar to the current commercial formulation after 24 weeks at the recommended storage temperature of 2 to 8°C.

Example 9 Stability of Formulations in Plastic Syringes.

A long-term study was conducted to monitor the stability of etanercept in PASS and SAS formulations at etanercept concentrations of 50 mg/mL in silicone oil-free COP pre-filled plastic syringe systems compared to siliconized glass pre-filled syringes. Stability was assessed at 1 ml fills in various syringe systems using SE-HPLC, pH and particulate matter assessment (HIAC) after storage at 4, 25 and 40°C. Protein concentration was tested only at time zero.

Materials: The drug used in this study was etanercept in TMS (10 mM Tris buffer, 4% mannitol, 1% sucrose) at a concentration of 25 mg/mL. The base volume used for the SAS formulation was titrated to pH 6.3. The material was ultrafiltered to ~50 mg/ml etanercept, then diafiltered into 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 a concentration of etanercept 50 mg/ml.

All formulations were manually filled into 1 ml glass syringes or 1 ml COP plastic syringes containing no silicone oil (COP A and COP B) to a volume of 1 ml and then stoppered using a vacuum stopper.

Methods: pH was measured using a Mettler Toledo SevenEasy pH meter in combination with a Mettler InLab MicroProbe. Before measurements, the samples were heated to room temperature. Protein concentration measurements based on absorbance at 280 nm for all samples were performed at room temperature using the Nano Drop system. Exclusive HPLC was performed on an Agilent 1100 HPLC with Chromeleon 7.2 software. Analysis of particles invisible to the naked eye was performed using a HIAC/Royco HACH particle counter system equipped with an HRLD-150 laser and Pharm Spec software. All samples were diluted with PASS formulation buffer to 25 mg/mL. Samples were thoroughly mixed, uncapped, and degassed for 2 hours at 75 torr prior to analysis. Four samples of 1.0 ml each (without tare volume) were analyzed, with the first sample discarded and averaging of the remaining three samples. Data for particle sizes of 2, 5, 10 and 25 µm were collected at all time points. Results take into account dilution and are presented as cumulative occurrence per ml.

Results and Discussion: Stability in silicone-free plastic syringes is similar to that in glass siliconized syringes. Protein concentration in all formulations was tested at time zero. The pH for all formulations was measured at time zero and after 12 weeks at 40°C and 24 weeks at 4 and 25°C. No change was observed with storage time or temperature, and all samples met the acceptance criteria for a pH of ±0.2 pH units from the target pH of 6.3. The results for protein concentration and measured pH values for all samples can be found in Table 1. 31.

Table 31

Protein concentration and pH results

Sample Concentration (mg/ml) pH value, t=0 pH, 24 weeks, 4°C pH, 24 weeks, 25°C pH value, 12 weeks, 40°C PASS_Glass 50.9 6, 3 6, 4 6, 4 6, 4 SAS_Glass 52.3 6, 3 6, 4 6, 4 6, 3 PASS_COP_A 51.1 6, 3 6, 4 6, 4 6, 3 SAS_COP_A 51.9 6, 3 6, 4 6, 4 6, 3 PASS_COP_B 51.1 6, 4 6, 4 6, 4 6, 3 SAS_COP_B 52.2 6, 3 6, 4 6, 4 6, 3

SE-HPLC was performed to control the levels of aggregation depending on the parameters of the composition, time and temperature. Peak B represents the amount of high molecular weight fragments (aggregates) formed. The results showed no difference in peak B between glass syringes and COP plastic syringes containing no silicone oil (Tables 32-34). Peak B represents the total number of aggregates detected by SE-HPLC for these formulations. All samples remained acceptable (peak B <6%) after 24 weeks of storage at 4 and 25°C.

Table 32

Peak B analysis by SEC, % of total, 4°C

Sample t=0 weeks t=4 weeks t=8 weeks t=12 weeks t=24 weeks PASS_Glass 2.9 thirty 3, 1 3, 1 3, 1 SAS_Glass thirty 3, 1 3.2 3.2 3, 1 PASS_COP_A 2.9 thirty 3, 1 3, 1 3, 1 SAS_COP_A thirty 3, 1 3.2 3.2 3.2 PASS_COP_B thirty 3, 1 3.2 3.2 3.3 SAS_COP_B thirty 3, 1 3.2 3.2 3.2 Table 33

Peak B analysis by SEC, % of total, 25°C

Sample t=0 t=2 t=4 t=8 t=12 t=24 weeks weeks weeks weeks weeks weeks PASS Glass 2, 9 thirty 3, 4 3, 9 4.4 5, 5 S.A.S Glass thirty 3, 1 3, 6 4.1 4.5 5, 6

-

Claims (17)

PASS_COP_A 2.9 3.1 3.4 3.9 4.4 5.4 SAS_COP_A 3.0 3.2 3.5 4.1 4.6 5.8 PASS_COP_B 3.0 3.2 3.6 4.1 4.6 5.5 SAS COP B 3.0 3.1 3.5 4.0 4.5 5.7 Table 34 Peak B analysis by SEC, % of total, 40°C t=0 t=12 Sample weeks t=2 weeks t=4 weeks t=8 weeks weeks PASS_Glass 2, 9 6, 4 11.1 17.2 23.0 SAS_Glass thirty 6, 1 9, 8 15, 4 21.0 PASS_COP_A 2, 9 6, 5 9, 9 16, 2 21.4 SAS_COP_A thirty 6, 3 9, 9 14.9 18.6 PASS_COP_B thirty 6, 4 10.7 17.8 19.6 SAS_COP_B thirty 6, 2 100 17.1 22.4 Particles invisible to the naked eye were monitored by chiaroscuro particle counting (HIAC). Results for formulations placed in BD glass syringes were in line with accumulated PFS data, while particles not visible to the naked eye were reduced in plastic syringes containing no silicone oil (Table 35). Table 35 Particles/ml (chiaroscuro mode), 4°С 2 µm 10 µm 25 µm t=0 t=24 t=0 t=24 t=0 t=24 weeks weeks weeks weeks weeks weeks PASS_Glass 10090 15994 468 726 14 2 SAS_Glass 12240 13340 1183 505 18 5 PASS_COP_A 93 130 2 6 0 0 SAS_COP_A thirty 74 2 2 0 0 PASS_COP_B 81 204 3 4 0 2 SAS_COP_B 62 237 2 8 0 1 Conclusion: The long-term stability of the SAS formulation at 50 mg/mL etanercept and the current commercial formulation based on etanercept stored in siliconized glass syringes and silicone oil-free COP syringes was evaluated at 4, 25, and 40°C. SE-HPLC showed no significant differences between formulations based on syringe type at 24 weeks. No pH shift was detected and all formulations remained within acceptable ranges. The number of particles invisible to the naked eye was reduced in COP plastic syringes containing no silicone oil and was consistent with the accumulated PFS results when stored in glass syringes. The results of the study showed that the SAS formulation at etanercept concentration of 50 mg/ml was stable and similar to the current commercial formulation after 24 weeks of storage at the recommended temperature of 2 to 8°C in different types of syringes. CLAIM 1. A pharmaceutical composition containing 75 to 150 mM NaCl, 5 to 100 mM arginine, 0.5 to 2% (w/v) sucrose, and 40 to 100 mg/mL etanercept, wherein the pharmaceutical composition contains less 2.0 mM total buffer other than etanercept and the pH of the composition is between 6.1 and 6.5. 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains less than 1.5 mM of a total buffer other than etanercept. 3. Pharmaceutical composition according to claim 2, where: a) the pharmaceutical composition contains less than 1.0 mM of a total buffer agent other than etanercept; b) the pharmaceutical composition contains less than 0.5 mM of a total buffer other than etanercept; c) the pharmaceutical composition contains less than 0.25 mM of a total buffer other than etanercept; or d) the pharmaceutical composition contains 0.1 mM or less of a total buffer other than etanercept. - 24 041785 4. The pharmaceutical composition according to claim 1, wherein the arginine is L-arginine. 5. Pharmaceutical composition according to claim 4, where: a) L-arginine is L-arginine hydrochloride or b) L-arginine is L-arginine in base form. 6. The pharmaceutical composition of claim 1, wherein the pH of the pharmaceutical composition is between 6.1 and 6.5 when stored at controlled room temperature (CRT) for 2 weeks. 7. The pharmaceutical composition of claim 6 wherein the pH of the pharmaceutical composition is from about 6.2 to about 6.3 when stored at controlled room temperature (CRT) for 2 weeks. 8. Pharmaceutical composition according to claim 1, where: a) the pH of the pharmaceutical composition is from 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 high molecular weight form, as determined using size exclusion chromatography; b) the pH of the specified pharmaceutical composition is from about 6.2 to about 6.5 for at least two weeks during the specified storage at about 25°C; or c) the pH of said pharmaceutical composition is from about 6.2 to about 6.4 for at least two weeks during said storage at about 25°C; or d) less than 28% of the total etanercept is in misfolded form as determined by hydrophobic interaction chromatography after two weeks of storage at about 25°C. 9. Pharmaceutical composition according to claim 1, where: a) the pharmaceutical composition is characterized by an osmolality of from about 180 to about 420 mOsm; b) the pharmaceutical composition has an osmolality of about 250 to about 350 mOsm; c) the pharmaceutical composition has an osmolality of about 290 to about 310 mOsm; or d) the pharmaceutical composition has an osmolality of about 300 to about 310 mOsm. 10. A pharmaceutical composition according to 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. 11. Pharmaceutical composition according to claim 1, additionally containing polysorbate 20. 12. A pharmaceutical composition according to claim 11 containing polysorbate 20 (w/v) at a concentration of from about 0.001 to about 0.1%. 13. A pharmaceutical composition according to claim 12 containing polysorbate 20 (w/v) at a concentration of about 0.005%, about 0.01%, or about 0.015%. 14. A process for preparing an etanercept pharmaceutical composition having a pH of 6.1 to 6.5, comprising replacing an etanercept formulation containing a buffer other than etanercept with a solution without any additional buffer, thereby obtaining a pharmaceutical composition containing from 40 to 100 mg/ml of etanercept and less than 2.0 mM of a total buffer agent other than etanercept, where each of the etanercept-based composition containing a buffer agent other than etanercept and a solution without any or an additional buffer agent, characterized by a pH of 6.1 to 6.5. 15. The method according to claim 14: a) where diafiltration is used in the replacement step; b) where the solution without any additional buffering agent is isotonic; c) where the solution without any additional buffering agent contains sucrose, arginine and NaCl; d) further comprising the step of filtering the pharmaceutical composition; or e) further comprising the step of separating the pharmaceutical composition into aliquots in the form of a finished dosage form. 16. The method according to claim 14, where the solution without any additional buffer contains sucrose, arginine and NaCl, where the concentration of NaCl is from 75 to 150 mm, the concentration of arginine is from 5 to 100 mm and the concentration of sucrose is from 0.5 up to 2% (w/v). 17. The method of claim 14, wherein the solution without any additional buffering agent consists of about 120 mM NaCl, about 25 mM arginine, about 1% (w/v) sucrose and water. -