Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

• the invention relates to the formulation of pharmaceutical compositions of etanercept.
• the invention also relates to methods of removing buffer and of formulating pharmaceutical compositions of etanercept
• Formulation of a protein drug can present many challenges for the pharmaceutical scientist, A formulation must be found that stabilizes the protein drug and makes it resistant to degradation by proteolysis, aggregation, misfolding, etc. Especially for- engineered, proteins that differ in substantial .respects to known proteins, finding appropriate stability conditions can. be challenging. It- is also desirable to have the protein drug be in a format that is convenient for the patient. Desired properties include stability at ambient and refrigerated temperatures: suitability for long term storage, appropriate dosing times and volumes; and minimization of discomfort upon administration.
• Etanercept is a dimerie fusion protein consisting of the extracellular iigand-binding portion of the human 75 kilodalton (p75) tumor necrosis feetor receptor (TNFR) linked to the Fc portion of human IgGl ,
• the Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CHI domain of IgGl. Whe expressed in mammalia ceils, it forms a homodimeric complex with two domains of the TNF receptor. Thus, it is an artificial protein that is different from both antibodies and soluble TNF receptors, and therefore subject to different degradation pathways than either.
• Etanercept is commercially available as EN B EL* (Amgen inc., Thousand Oaks, CA) . and. is approved to treat moderately to severely active rheumatoid arthritis, moderately to severely active polyarticular juvenile idiopathic arthritis (JIA) in patients ages two and older, chronic moderate to severe plaque psoriasis (PsO) in adults, psoriatic arthritis (PsA) in adults, and active ankylosing spondylitis (AS). Etanercept was first available in a lyopbilized formulation to be reconstituted immediately before injection.
• EN B EL* Amgen inc., Thousand Oaks, CA
• JIA polyarticular juvenile idiopathic arthritis
• PsO chronic moderate to severe plaque psoriasis
• PsA psoriatic arthritis
• AS active ankylosing spondylitis
• formulation is 1 mM Tris i!Q, 4% mannitol, 1 % sucrose, pH 7.4 at about 25 mg/ nL.
• An exemplary liquid formulation consists of 50 mg/ ' mL etanercept, 25 m
• phosphate buffer 25 mM L-arginine hydrochloride, 100 mM NaCl, 1% Sucrose at pH 6.3 in water.
• the invention provides pharmaceutical compositions containing etanercept that are stable and can. be conveniently stored as a liqu d at controlled room temperature (CRT) for extended periods of time, even in the absence of an additional buffering agent.
• CRT controlled room temperature
• compositions of the in vention are injected into subjects, they also demonstrate significantly reduced injection pain as compared to the commercially available prior art formulation. Thus these pharmaceutical compositions are more convenien and advantageous for patients.
• Another aspect of the invention provides methods of formulating pharmaceutical preparations of etanercept at a desired pH but in the absence of additional buffering agent in the final formulation.
• the invention provides a pharmaceutical compositio comprising etanercept, NaCl, arginine, and sucrose, wherein the pharmaceutical composition has essentially no 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 ihe pH between 6.1 and
• the etanercept concentration is between 0 mg/iriL and 10 .
• the pharmaceutical composition is Isotonic,
• the pharmaceutical composition contains: between 20 mM and 150 mM NaCl; between 5 mM and 100 mM arginine; and between 0.5% and 2% ( /v) sucrose.
• 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 poiysorbate 20 at a concentration (w/v) of between 0.001% and 0.1 %. In. nother embodiment, the surfactant is polysorbate 80 at a concentration (w/v) of between 0.001 % and 0,1 %. I another embodiment, the surfactant is poloxamer 188 at .a concentration (w/v) of between 0.01 to 0.3%.
• the pharmaceutical composition maintains a pH of between 5.8 and 6.7 for at least two weeks when stored at approximately 25° C, and wherein less than 6% of the totai etanercept is aggregated m a high molecular weight form as assessed using size exclusion chromatography.
• the pharmaceutical composition maintains a pH of between about 6, 1 and about 6,5.
• less than 28% of the total amount of ⁇ etanercept is in a misfolded form as assessed using hydrophobic interaction chromatograph . in another
• the pharmaceutical composition consists essentially of 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 consists essentially of about 40-100 mg/mL etanercept, about 120 mM NaCi, about 25 mM arginine, about 1% sucrose, about 0.01% polysorbate 20. and water.
• the present invention provides a method of formulating a
• composition of etanercept to remove an additional buffering agent and maintain pH from 6.1 to 6.5 comprising formulating the etanercept formulation in a formulation comprising an additional buffering agent at between pH 6.1 and 6.5. and exchanging the formulation comprising an additional bufferin agent against a formulation tha does not comprise an additional buffering agent and is between pH 5.6 aid 6.5, and collecting the resulting pharmaceutical formulation.
• the exchange step uses diafiltration.
• the formulation that does not comprise an additional buffering agent Is isotonic.
• tire formulation that does not comprise an additional buffering agent contains sucrose, arginine, and NaCI
• the formulation that does not comprise an additional buffering agent contains between 20 mM and 150 mM NaCI; between 5 mM and 100 mM arginine; and between 0.5% and 2% (w/v) sucrose.
• the formulation that does not comprise an additional buffering agent consists essentially of about 120 mM NaCL about 25 mM arginine, about 1% sucrose, and water.
• the method of formulating a pharmaceuti cal composition of etanercept further comprises adding polysorbate.
• the polysorbate is polysorbate 20 at a concentration (w/v) of between 0.001% and 0.1%.
• composition of etanercept further comprises filtering the pharmaceutical composition.
• the method of formulating a pharmaceutical composition of etanercept further comprises aliquoting the pharmaceutical composition into a drug product form.
• the present invention provides kit comprising a pharmaceutical composition of etanercept as described above in a drug product form and instructions for storage and use.
• the present invention provides a pharmaceutical composition comprising etanercept.
• etanercept NaCl, arginine, sucrose, a phosphate buffer and benzyl alcohol, wherein the pH of the composition is between 6.1 and 6.5.
• the benzyl alcohol is at a concentration (v/v) of between 0.1 % and 5.0%, I another embodiment, the concentration of benzyl alcohol is about 0.9%, in another embodiment, the pharmaceutical composition comprising etanercept further comprises polysorbate 20 at a concentration ( /v) of between 0.001 % and 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.004%.
• the pharmaceutical composition comprising etanercept consists essentiaiiy of etanercept at about 40-100 mg/mL, arginine at about 25 mM, sodium chloride at about 100 mM, sucrose at a concentration (w/v) of about 1%, phosphate buffer at about 25 mM, and benzyl alcohol at a concentration (v/v) of -about 0.9%, in another
• the pharmaceutical composition comprising etanercept consists essentiaiiy of etanercept at about 40-100 mg/mL, arginme at about 25 mM, sodium chloride at about 100 mM, sucrose at a concentration (w/v) of about 1%, phosphate buffer at about 25 mM, benzyl alcohol at a concentration ⁇ v/v ⁇ of about 0.9%, and polysorbate 20 at a concentration (w/v) of about 0.004%.
• the present invention provides a single-dose container containing a pharmaceutical composition comprising etanercept as described above.
• the pharmaceutical composition consists essentially of etanercept at about 40-1 0 mg/mL, arginine at about 25 mM, sodium chloride at about 100 mM, sucrose at a concentration .(w/v) of about 1 %, phosphate buffer at about 25 mM, and benzyl alcohol at a concentration (v/v) of about 0.9%
• the pharmaceutical composition consists essentially of etanercept at about 40-100 mg/mL, arginine at about 25 mM, sodium chloride at about 00 mM, sucrose at a concentration (w/v) of about I %, phosphate buffer at about 25 mM, benzyl alcohol at a concentration (v/v) of about 0.9%, and polysorbate 20 at a concentration (w/v) of about 0.004%.
• the single dose container is a vial, a syringe, or an autoinjector.
• the single-dose container contains an aqueous formulation consisting of etanercept at 50.0 mg/mL, sodium chloride at 120 mM, L-arginine at 25 mM, sucrose at 1.0% (w/v).
• the present invention provides method of preparing a single-dose container containing a pharmaceutical composition comprising etanercept as described above, compri sing filling the single-dose container with about a single dose of the pharmaceutical composition under sterile conditions.
• Figure 1 shows the percent. HMW (peak B) as detected by SEC for the etanercept stability assay of Example 3 ,
• Figure 2 shows the percent LMW as detected by dSEC for the etanercept stability assay of Example 3.
• Figure 3 shows the percent Peak 3 as detected by HIC for the etanercept stability assay of Example 3
• Figure 4 shows the percent Peak 3 as detected by HIC for the stainless steel cryo- vessei storage etanercept stability assay of Example 4.
• Figure 5 shows the percent LMW as detected by dSEC for the stainless steel cryo- vessel storage etanercept stability assay of Example 4.
• Figure 6 shows d e percent Peak B as detected by SEC for the stainless steel cryo- vessel storage etanercept stability assay of Example 4,
• Figure 7 shows the percent Peak B as detected by SEC for the freeze-tliaw etanercept stability assay of Example 4.
• Figure 9 shows the IJF/DF pool pH (A) and conductivity (B) stability at CRT in the assay of Example 6.
• Figure 10 shows the pH (A) and conductivity (B) stability of etanercept formulated in S AS solution in the assay of Example 6.
• the invention provides improved pharmaceutical compositions of etanercept.
• pharmaceutical composition is understood to refer to formulation of a polypeptide suitable for injection and or administration into a. patient in need thereof. More particularly, a pharmaceutical composition is substantially sterile and does not contain any agents that are unduly toxic or infectious to the recipient.
• Etanercept is produced by recombinant DN A technolog 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 kilodaitons (Physicians Desk Reference, 2002, Medical Economics
• the invention provides a pharmaceuti al composition comprising etanercept hut containing essentially no additional buffering, agent.
• 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 herein to provide all the needed buffering to maintain the pM between 6,1 and 6.5, and in particular at about 6.2 63, under the conditions described below,. As demonstrated below in Example 1, this pH range has been shown to be effective to maintain the desired stability characteristics of an etanercept formulation (less than 6% high molecule weight aggregates and less than 28% misfolded and clipped species).
• compositions according to the inventio comprise less than 2.0 mM total additional buffering agent, less than 1.5 mM total additional buffering agent, less than 1 .0 mM total additional buffering agent, less than 0.5 mM total additional buffering agent, less than 0.25 mM total additional buffering agent, less than 0.1 mM total additional buffering agent, or less than 0,05 mM of total additional buffering agent.
• additional buffering agents are used to maintain the pH in a desired range, often at concentrations of 5.0 mM or higher.
• additional buffering agents are hisiidu e, potassium phosphate, sodium or potassium citrate, ma!eic acid, ammonium acetate, .
• tris-(hydroxymefhyl)-aminomethane tris
• tris tris-(hydroxymefhyl)-aminomethane
• One common buffering agent is sodium phosphate as its buffering capacity is at or near pH 6.2
• Sodium phosphate is the buffering agent used in the current commercial liquid formulation of etanercept since its desired pH is 63, in the invention described herein, essentially no sodium phosphate is present in the pharmaceutical formulation of etanercept.
• the pH of the inventive pharmaceutical composition is maintained between 6.1 and 6.5, even alter extended storage.
• the pharmaceutical composition with essentially no additional buffering agent results in significantly less pain than the current buffered commercial formulation.
• phosphate is often chosen as a buffer for pharmaceutical compositions because of the close to neutral pH buffering capacity and belief that it is one of the less painful buffer components (as compared to, for example, citrate buffer), the instant inventors have determined thai phosphate buffer at around pH 6.3 does contribute to pain upon injection.
• formulation buffer * is a solution or buffer that does not itself contain etanercept but is used to make a formulation comprising etanercept.
• the etanercept concentration in tire pharmaceutical compositions of the invention is between about 40 mg/mL and about 200 mg/mL in an aqueous formulation (e.g., water as the solvent). More preferably, the etanercept concentration is between about 40 mg/mL and about 100 mg/mL, yet more preferably between about 40 mg mL and about 75 mg/mL, and optionally about 50 mg mL,
• the pharmaceutical compositions of the invention also contain argimne.
• Argimoe has been shown to make a substantial contributions stabilizing etanercept in a liquid formulation (see US Patent No. 7,648,702, incorporated herein by reference).
• Pharmaceutically appropriate forms of arginine are commercially available.
• L-arginine e.g., L-arginine HC1 or L - arginine base
• arginine is the arginine used for pharmaceutical formulations. It is understood thai within the pH range of 6.0 and 6.6, and in particular at a pH of about 6.2 - 6.3. arginine does not contribute meaningfully to the buffering capacity of a formulation.
• the concentration of arginine i the compositions of the invention are preferably .from about I mM to about 1 M, more preferably from about 10 mM to about 200 mM, or alternatively from about 5 mM. to about 100 mM, more preferably from abou 10 mM to about 100 mM. eve more preferably from about 15 mM to abou 75 mM, and yet more preferably at about 25 mM.
• the pharmaceutical composition comprises about 50 mg/mL to 75 mg mL etanercept and about .25 mM arginine, wherein the pharmaceutical composition has essentially no additional buffering agent, and the pH of the composition is between 6.0 and 6.6.
• the term "about” is understood to mean that there can be variation in the concentration of a component of the described formulation thai can be up to and including 10% of the given value. For example, if a formulation has abou t 30 mg/mL of a polypeptide, this is understood to mean that a formulation can ha ve between 9 to 11 mg/mL of the stated polypeptide,
• the phannaceutical compositions may contain additional exeipients, as long as those exeipients are not additional buffering agents, and in particular are not phosphate buffering agents.
• additional exeipients according to the invention include but are not limited to sugars/polyois such as: sucrose, lactose, glycerol, xylitol, sorbitol, mannitoi, maltose, inositol, trehalose, glucose: polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropyl metbyjceiiulose (HPMC),
• BSA bovine serum albumin
• HPMC hydroxypropyl metbyjceiiulose
• polyethyieneimine gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); nonaqueous solvents such as: polyhydrie alcohols, (e.g., PEG, ethylene glycol and glycerol) dimethy sulfoxide (D SO) and dimethylformamide (DMF); amino acids such as; proline, L- serine, alanine, glycine, lysine hydrochloride, sareosine and gamma-aminqb3 ⁇ 4rtync acid, and surfactants.
• PVP polyvinylpyrrolidone
• HEC hydroxyethylcellulose
• nonaqueous solvents such as: polyhydrie alcohols, (e.g., PEG, ethylene glycol and glycerol) dimethy sulfoxide (D SO) and dimethylformamide (DMF); amino acids such as; proline, L- serine, a
• the exeipients include aCl and or sucrose.
• NaCl may be present in the pharmaceutical composition at a concentration of .from about 5 mM to about 200 mM, more preferably between about 20 mM to abou 150 mM, even more preferably between about 80 mM to about 140 mM
• Sucrose may be added to a concentration of between about 0.5% to about 2% (w/v) sucrose, more preferably between about 0.8% to about 1.2% (w/v) sucrose, even more preferably at about 1% (w/v) sucrose.
• the osmolalit of a pharmaceutical composition is preferabl regulated in order to maximize the active ingredient's stability and also to minimize discomtbrtto the patient upon administration. It is generally preferred that a pharmaceutical composition be isotonic with serum, i.e. , having the same or similar osmolality, which is achieved by addition of tonicity modifier. Serum is approximately 300 +/- 50 milliosmolals per kilogram, thus it is
• the osmolality of an isotonic pharmaceutical composition will be from about 180 to about 420 milliosmolals. In some embodiments, the range will be. from about 250 to about 350 milliosmolals.
• a tonicity modifier is understood to be a molecule that contributes to the osmolality of a solution.
• tonicity modifiers suitable for modifying osmolality include, but are not limited to amino acids (e.g., arginme, cysteine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or saccharides (e.g., sucrose, glucose and mannitoi).
• the concentration of the tonicity modifier in the formulation is preferably between about 1 m to I , more preferably about 10 mM to about 200 mM. In some embodiments, the concentrations of NaCl and sucrose are adjusted to generate a pharmaceutical composition that is isotonic.
• the pharmace tical composition contains about 40-100 rng/raL etanereept, about 120 mM NaCl, about 25 mM arginine, about 1 % sucrose, and water
• the pharmaceutical composition can consist essentially of about 50-100 mg/mL e anereept, about 120 mM NaCl, about 25 mM argrnme, about 1% sucrose, about 0.01% polysorbate 20, and water.
• the phannaceutical compositions of the invention. may include a surfactant.
• Surfactants are agents that reduce solution/surface induced stress.
• examples of surfactants are- polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 (e.g.. TWBEN-20® (Sigma-Aldrich, St. Louis, MO) or TWEBN-80® (Sigma- Aidrick St.. Louis, MO)), sodium dodecyl sulfate (SDS), polyoxyemylene copolymer, . poloxaniers, such as
• polysorbate 20 can be included in the pharmaceutical compositions at a .concentration (w/v) of between about 0.001% and about 0.03%, in particular embodiments illustrated below by- example, polysorbate 20 can be included in the pharmaceutical formulations at a concentration (w/v) of 0.01% or at about 0.004%.
• test containers which may be glass vials, glass syringes, plastic syringes, stainless steel vessels, or an manner of sterile device suitable for pharmaceutical compositions
• the pH is assessed at time 0, and then at indicated times as appropriate.
• the testing conditions will anticipate need for storag of the test containers
• the formulations of the invention are able to maintain the desired pH under controlled room temperature (CRT) for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, and at least 24 weeks.
• CRT is defined by the USP, and has a temperature maintained thermostatically that
• the pharmaceutical compositions of the invention exhibit particular quality attributes.
• the testing of these quality attributes is also described below by way of example.
• the pharmaceutical compositions of the invention contain less than 6% of the total etanercept aggregated in a hi gh molecular weight form as assessed using size exclusion chromatography.
• the pharmaceutical compositions of the in vention contain less than 28% of the total amount of etanercept is in a roisfalded form as assessed using hydrophobic interactio chromatography.
• the pharmaceutical composition of the invention are capable of remaining stable by maintaining pH and/or other noted quality attributes (minimum high molecular weight forms and minimum misfolded forms) for the following temperatures and extended dme 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) for up to 1 freeze/thaw cycle, up to 2 freeze/thaw cycles, up to 3
• pH and/or other noted quality attributes minimum high molecular weight forms and minimum misfolded forms
• freeze/thaw cycles and up to 5 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 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 4G° C (accelerated stability testing) for at least 2 weeks,
• compositions of the invention also exhibit the surprising result of reduced pain upo injection into a subject.
• This property can be assessed using the Visual Analog Scale (VAS) that has been validated by Gallagher el al, 2002, Am, J. Em. Med. v20; i4: 287-290. Trained health professionals administer the drug via injection, and within 30 seconds after each injection, subjects assessed their level of injection pain using a 100 mm Visual Analog Scale (VAS). A difference of 13 to 16 mm on the VAS is considered to be clinically meaningful.
• VAS Visual Analog Scale
• etanercept is expressed recombinant! y in CHO cells and secreted into, the medium.
• the medium is collected, filtered, and purified using, for example, various chromatography techniques.
• protein A can be used to purify Fc domain containing polypeptides such as etanercept, and is advantageous s a first processing step.
• polypeptide purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETTM, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and any combination of purification techniques known or yet to discovered. Examples of useful production and purification techniques can be found in US Patent Nos. 7,294,481 ( Fung),
• the invention also provides a method of formulating a pharmaceutical composition of etanercept to remove buffer and maintain pH at a target range, comprising formulating the etanercept in a buffered formulation in the target range, and exxhanging the buffered
• the method provides formulating a
• composition of etanercept to remove buffer and maintain pH from 6.0 to 6.6 comprising formulating the etanercept formulation in a buffered formulation at between pH 6.0 to 6.6, and exchanging the buffered formulation against an unbuffered formulation mat is between pH 5.6 and 6.5, and collecting the resulting pharmaceutical formulation, to order to achieve an unbuffered composition of etanercept that maintains its pH from 6,1 to 6.5, it is important to ensure that the pH of both the starting buffered etanercept formulation and the unbuffered formulation is calibrated.
• the starting buffered etanercept formulation is at H 7.2, it will be adjusted with a strong acid, such as HC1, to withi the range 6,1 to 6.5.
• the unbuffered formulation that is used for exchange should be titrated to between pH 5.6 and 6.5. Because the unbuffered formulation used for exchange has no buffering agent, care should be used d ring titration,
• Dialysis makes use of selective diffusion through a semi-permeable membrane ' to remove unwanted smaller molecules from a larger protein formulation.
• equ librations are done serially until a desired fold reduction in the concentration of an unwanted molecule is achieved. For example, three serial equilibrations, each at or greater than 100-fold dilution, can be used to achieve a concentration reduction of 1,000,000- told, or greater.
• Ultrafiltration and diafl!tration are similar to dialysis in that they use a semi-permeable membrane. But unlike the passive diffusion of dialysis, ultrafiltration and diafiltration involves forcing solutions through the membrane using various techniques. Pressure and centrifugation are typically used.
• Still another method of buffer exchange can he performed using gel filtratio or size exclusion chromatography.
• chromatographic techni ues that also can be used to achieve buffer exchange that are well within the skill of those in the art such as ion exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography.
• the methods of the invention include collecting the resulting pharmaceutical formulation. At this point, essentialiy all buffer has been removed, but the pH is still maintained at the desired levels. For a pharmaceutical composition containing etanercept, the pH is maintained at between 6.0 and 6.6.
• the pharmaceutical formulation may be further treated as necessary .
• a surfactant can be added, in another example, if desired to remove particles, tlie pharmaceutical composition can be filtered.
• the methods of the invention also include aliqitoting the pharmaceutical compositions into a drug product, form. Such drug product forms are distributed for final use by patients or health care providers.
• compositions of tins invention are particularl useful for parenteral
• administration i.e., subcutaneousl , intramuscularly, intravenously, intraperitoneal,
• compositions for injection may be presented in unit dosage form, e.g.. in ampoules or in multi-dose containers.
• the pharmaceutical compositions may, if desired, be presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient, in one embodiment the dispenser device can comprise a syringe having a single dose of the liquid formulation ready for injection.
• the pharmaceutical composition is aliquoted into a cassette component for use with a reusable autoinjector.
• the pharmaceutical composiiions can be provided packaged in or with an on-body injector device, in still another embodiment, the phanrtaceuiieal compositions can be aliquoted into a drug product form suitable for needleless injection device.
• the pharmaceutical composition can also be aliquoted into a format suitable as a depot preparation.
• Such long acting formulations may be administered by implantation (For example subeutaneously or intramuscularly) or by intramuscular injection.
• the formulations may be modified with suitable polymeric or hydrophobic materials (for example as an emulsion in an. acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• the present in vention is directed to a kit. or container, which contains the pharmaceutical composition of the invention
• the kit can also be accompanied by instructions for the storage and use of the pharmaceutical compositions.
• the container can be, for example, a single-use container, i.e., a container tha holds one dose formulation of the present invention. It is understood that a single-use container might contain a single dose plus enough extr to ensure that a. full single dose can be administered to a patient from the
• containers suitable for use in certain aspects of the present invention include vials, syringes, arid auto-injectors.
• suitable auto-injectors include those found in US Pat. Nos. 8,177,749, 8,052,645, and 8,920,374, in. US Pat App. Ser. Nos. 12/993163, 13/269750, 13/454531, 14/112479,
• the etanercept-contammg compositions and formulations of the present invention can be used in the treatment of patients with conditions that respond to treatment with etanercept.
• conditions that respond to treatment with etanercept include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and psoriasis.
• Methods of treating patients with etanercept are described in, for .example, US Pat, Nos. 7,915,225, 8,119,605, 8,410,060, 8,722,631, aid 8,119,604, each of which is incorporated herein by reference in its entirety.
• a 1% stock solution of poiysorbate 20 was prepared fresh and spiked into all formulation to a final concentration of 0.004%.
• AH formulations were manually filled into 1 raL long BD glass syringes to a volume of 0.5 mL and then stoppered using an ASPll vacuuming stoppering unit.
• the high concentration formulation 1 OC SAST J QONaCl stored at 25° C and 40° C began to show an increase in high molecular weight aggregates when analyzed using size exclusion chromatography, but performed similarly to the current commercial formulation a 4° C
• the FASST+BeOH which is the current commercial formulation modified by the addition of 0.004% polysorbate 20 and 0.9% benzyl alcohol ) performed similarly to the current commercial formulation at both 4° C and 25° C but experienced an. increase in high molecular weight species by SE-HPLC at later time points when stored at the elevated temperature of 40° C.
• the 50_S AST 1 OONaCl formulation maintained levels of high and low molecular weight species that were comparable to the current, commercial formulation at ail temperatures, even in the absence of phosphate buffer.
• Test formulations (detailed below in Table 9) were administered by a trained healthcare professional in 6 unique sequences with 8 subjects randomized to each sequence. Injections were administered in each quadrant of the anterior abdominal wall and administered approximately 1 hour apart, Withi 30 seconds after each injection, subjects assessed their level of injection pain using a 100 mm Visual Analog Scale (VAS). Adverse events were collected from the beginning of the first injection through 30 days alter the first ' injection. Safety follow- up phone calls were conducted on day 2 (24 hours after the sixth injection) and day 31 ( ⁇ 2 days). Table 9: T ested Formulations
• Negative pain 1.0 10 mM sodium acetate, 9% (w/v) sucrose,
• Test formulation 1.0 100 mM sodium chloride, 25 mM L ⁇
• fiiOS&J Summary statistics (mean, SD, standard .error [SB], median, minimum, maxi mum) were calculated for VAS scores by solution.
• VAS scores were analyzed using an analysis of variance (ANOVA) model, which included sequence, solution, and period as independent variables, and subject, within sequence as a random effect. No adjustment was made for multiple comparisons.
• ANOVA analysis of variance
• SAST 120NaCl was prepared by diluting the 75 mg mL SAS material using a concentrated NaCI stock solution to achieve a final concentration of 120 mM NaCL
• a 1 % stock solutio of polysorbaie 20 was prepared fresh and spiked into ail formulation to a final concentration of 0.01 %.
• AH formulations were manually filled into 1 ml, long BD glass syringes to a volume of 1 ml, and then stoppered using an ASPU vacuuming stoppering unit.
• HPLC Hydrophobic interaction.
• HPLC was run on an Agilent 1 100 HPLC with Chromeleon 7.2 software at an absorbance of 215 nm.
• Denatured size exclusion HPLC was run on an.
• Sub-visible particle analysis was performed using a BACH HlAC/Royco particle counter system equipped with an HRLD-150 laser and Pharm Spec software.. All samples were diluted with PASS formulation buffer to 25 mg/rnL. Samples were thoroughly mixed, uncapped and degassed for 2 hours at 75 torr prior to analysis. Pour (4) sips of 1.0 mL each (no tare volume) were performed, with the first sip discarded and the remaining three sips averaged. Data for particle sizes 2. 5, 0, and 25 ⁇ was collected at all time points. The results account for the dilution and are reported as cumulative counts per milliliter.
• Osmolality was tested at time ero only. T3 ⁇ 4e osmolality results for all samples can be found in Table 14, AM formulations were at their target osmolality. Due to differences in buffer and excipient levels, the osmolality was not expected to be the same across the various formulations.
• Peak B is the amount of high molecular weight species (aggregate) that forms. Results showed no differences in Peak B between the PASST control and the buffer-less formulations at 4° C and 25° C, with minor differences being observed after twelve weeks at 40° C ( Figure 1). Peak B represents the total aggregate detected by SB-HPLC for these formulations. All samples remained acceptable (Peak B ⁇ 6%,) after 52 weeks of storage at 4° C, 24 weeks of storage at 25° C, and after twel ve weeks, of storage at 40 3 ⁇ 4 C.
• Example 4 Freeze/thaw and long term stability of top reformulation candidates in stainless steel containers
• Etanercept 25 mg/mL, in I MS (10 mM Iris HC1, % mannitol, 1 % sucrose, pH 7.4); SAS l OONaCl solution (100 mM HaCL 25 mM L-arginine FICL 1 % sucrose, pH 6.3) for dialysis; PASS buffer (25 mM Phosphate, 100 mM NaO, 25 mM L-arginine HO, 1% sucrose, pH 6.3); 1.0.000 MWCO centriprcps; 3-12 mL SHde-A ⁇ Lyzer dialysis ; cassettes, 10,000 MWCO; a Mettler Toledo MP220 pH .meter and Mettler Toledo InLab MicroProbe. Methods: For the UF/DF example, 25 mg mL etanercept in IMS was concentrated to
• the material was then dialyzed using 10,000 M WCO slide-a-lyzer dialysis cassettes, 9.5mL of 50 mg/mL etanercept in I MS was added to the cassette and exchanged against .1 QOOirsL of SAS 100. Three exchanges were performed to achieve a 1 ,000,000 fold exchange. The first exchange occurred at 5pm on Day 1 and went overnight The second 1,000 mL exchange was at 8:30am on Day 2. The third and final exchange was at 12:30pm on Day 2. At 5:00pm on Day 2 the protein was removed from die dialysis cassette (11 mL removed) and the pH was measured on the same Mettler Toledo MP220 pH meter. The measured pH was 6,98.
• the SAS formulation solutio is composed of 120 mM sod um chloride, 25 mM L-argimne, 1% sucrose, pH 6.3.
• An SAS formulation solution was titrated to pH 6.3 using 1.0 N NaOH.
• the volume of titrant required to reach the specific pH range was 4.4 pL/L SAS formulation solution.
• the pH of the permeate remained close to the pH of WFi rather than the pH of the SAS formulation soiution. Without being bound to a particular theory, this is believed to he due to the low buffering capacity of the SAS formulation solution.
• the expected range for conductivity of the permeate following membrane equilibration usin the range of the SAS formulation solution preparation is 12-16 mS/cm.
• lOOSlJ AE intermediate Pool Starting Material Prior to transferring the AEX intermediate pool into the retentate tank of an UF/DF tank, the pool was conditioned using 2 M HC1 to a target pH of 6,3 (acceptable range 6.2-6.4). The volume of titrant required to reach the specific pH. range was approximately 2.8 rnL/L AEX intermediate pool.
• Runs 4 through 7 were only measured for pH, conductivity, osmolality, and protein concentration in order to determine impact of formulation solution pH and load H impact U F/DF pool pH.
• the conditioned AEX intermediate pool can be held for up to 52.6 hours at controlled room temperature (CRT).
• CRT controlled room temperature
• the final UF/DF SAS pool generated using AEX intermediate pool as the starting material, can be held for up to 96.3 hours at CRT.
• the pH and conductivity during the hold are shown in Figure 9 A and B. Over the 96.3 hour hold, the pH and conductivity remain within acceptable limits.
• PASS DS Intermediate Pool Starting Materia! .No conditioning is required prior to transferring the PASS DS intermediate pool into the UF/DF retentate tank because the PASS DS intermediate pool is already within the acceptable pH range.
• the starting material is 50 rng/mL PASS formulated Enbrel DS, the pool does not need to be concentrated to 50 g/L because it is alread at the correct concentration to perform diafiltration.
• Results The product quality results for the final SAS UF DF pool, generated using PASS DS intermediate pool as the starting material, are shown i Table 21.
• the step yield for Run 1 was o utside of the acceptance criteria; however, it was most likely an artifact of bench- scale processing and considered not significant to the conclusions of the study.
• the final SAS UF/DF pool also met acceptance criteria for product quality using SEC and HiC analysis, as described above.
• the PASS pool does not require conditioning prior to UF DF processing with SAS solution because this intermediate pool is already at the target pM (6.3).
• a pool hold study was not performed for this intermediate pool because the conditions of the pool were unchanged from Enbrel PASS DS, The pool can be held for up to 96 hours at 25° C
• the final UF/DF SAS pool, generated using PASS DS intermediate pool as the startin material can be held for up to 96.3 hours at CRT.
• the pH and conductivity during the hold are shown in Figure 9 and B. Over the 96.3 hour hold, the pH and conductivity remain within acceptable limits.
• the SAS formulation solution can be held for up to 28 days at CR T.
• the pH and conductivity are shown in Figure 10 A and B. Over the 42 day hold in small scale stainless steel stability chambers with very small headspace, the SAS formulation solution is demonstrated to maintain a pH within 5.6 to 6.5. There was precipitation observed at the 35 day and 42 day time points. The 21 day time point measurement of 5.09 appears to be ait outlier due to the fact thai the subsequent time points are within the proposed acceptance criteria.
• the final UP/DF unit operation can produce 50 g L SAS formulation produc and achieve consistent product qualit - compared to the current commercial PASS formulation product under the following process recommendations: 1 ) utilizing either AEX intermediate pool, or PASS DS intermediate pool, as the starting material 2) the SAS solution can he held for at least 28 days at CRT and maintain a pH of 5.6 to 6.5, 3) the conditioned AEX intermediate pool can be held at CRT for at least 52.6 hours and maintain a pH of 6.3 ⁇ 0.1». and 4) the SAS formulated UF/DF pool can be held at CRT for at least 96.3 hours and maintain a pH of 6.1 to 6.5 and a conductivity of 10 to 14 mS/cm.
• Osmolality was measured using The Advanced Osmometer Model 3900. Each measurement was performed using 250 uL of sample and 290 osmolality standards were tested to ensure the system was operating properly. Size exclusion HPLC was run on an Agilent 1 1 GO HPLC with Chrome!eon 7.2 software.
• Table 24 SEC aggregate/HMW levels, % of total, 40° C
• Example 8 Stability of formulations with various levels of polysorbate 20
• SE ⁇ HP:LC was performed to monitor aggregation levels as a junction of formulation condition, time and temperature.
• Peak B is the amount of high molecular weight species (aggregate) that forms. Results showed no differences in Peak B between the PASS control and tbe bufferless formulations at all temperatures at their respective protein concentrations (Table 27-29). Peak B represents the total aggregate detected by SE-HPLC for these formulations. All 50 mg mL samples remained acceptable (Peak B ⁇ 6%,) after 24 weeks of storage at 4° € and 25° C, and after 2 weeks of storage at 40° C.
• Example 9 Stability of formulations in plastic syringes
• SE-HPLC was performed to monitor aggregation levels as a function of formulation condition, time and temperature. Peak is the amount of high molecular weight species (aggregate) that forms. Results showed no differences in Peak B between the glass syringes and the COP plastic silicone oil free syringes (Table 32-34). Peak B represents the total aggregate detected by SE-HPLC for these formulations. All samples remained acceptable (Peak B ⁇ 6% j after 24 weeks of storage at 4°Car»d 25°C.

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