WO2015138844A1 - Procédés pour la production de formulations de protéines cytokine chimériques stables dans des récipients du type à soufflage/remplissage/fermeture - Google Patents

Procédés pour la production de formulations de protéines cytokine chimériques stables dans des récipients du type à soufflage/remplissage/fermeture Download PDF

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Publication number
WO2015138844A1
WO2015138844A1 PCT/US2015/020357 US2015020357W WO2015138844A1 WO 2015138844 A1 WO2015138844 A1 WO 2015138844A1 US 2015020357 W US2015020357 W US 2015020357W WO 2015138844 A1 WO2015138844 A1 WO 2015138844A1
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WIPO (PCT)
Prior art keywords
formulation
bfs
seq
container
chimeric cytokine
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Application number
PCT/US2015/020357
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English (en)
Inventor
Gregory Zarbis-Papastoitsis
Patricia LOWDEN
Emily Belcher SCHIRMER
Waiken K. Wong
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Eleven Biotherapeutics, Inc.
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Application filed by Eleven Biotherapeutics, Inc. filed Critical Eleven Biotherapeutics, Inc.
Publication of WO2015138844A1 publication Critical patent/WO2015138844A1/fr

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    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2006—IL-1
    • 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
    • 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
    • A61K9/0048—Eye, e.g. artificial tears

Definitions

  • the invention relates to methods of producing a therapeutic biologic.
  • plastic ampuls offer several advantages, such as, convenience, and being simple to use and child-safe.
  • the high temperatures at which such ampuls are filled are a disadvantage for biologies which are generally sensitive to thermal damage.
  • Plastic ampuls can be manufactured using blow-fill-seal (BFS) technology.
  • BFS blow-fill-seal
  • the process of packaging formulations using BFS technology generally involves plastic extrusion, molding the extruded plastic to form BFS containers (which may also be referred to as ampuls, vials, or units), aseptic filling of the BFS containers with the formulation, and hermetic sealing of the filled containers, with these steps performed in sequence.
  • BFS blow-fill-seal
  • the material used to form BFS containers is typically supplied asgranules of a thermoplastic resin, for example, low-density polyethylene or polypropylene.
  • a thermoplastic resin for example, low-density polyethylene or polypropylene.
  • the granules may be melted at temperatures above 160°C.
  • the plastic is molded into the desired container shape, filled with formulation, and hermetically sealed.
  • the BFS containers may be partially cooled during and after the molding step and before the filling step. For example, cooling water may be circulated around the mold, which may help to lower the temperature of the BFS containers.
  • this cooling process is limited by the relatively short contact time between the BFS container and the mold (typically only about 10 seconds), and by the need to keep the temperature of the BFS containers sufficiently high, to ensure that an appropriate seal can be made at the end of the filling process.
  • BFS processing is not generally suitable to produce packaged therapeutic protein-containing formulations with acceptable stability. It is expected that because the formulation is necessarily exposed to heat from the containers during the filling process, therapeutic proteins can lose stability, e.g., as indicated by formation of particulates, aggregates and modified (e.g., oxidized) forms.
  • Applicants have discovered methods for successful production of a chimeric cytokine formulation in BFS containers, such that the formulation retains stability immediately following BFS processing and after extended storage.
  • methods and apparatus for packaging a formulation comprising a chimeric cytokine protein using a BFS process are Featured herein.
  • Described herein are methods for BFS processing involving temperature control of the transfer system through which the formulation described herein passes prior to dispensation into BFS containers that permit the production of BFS containers containing stable formulations. Also described herein are stable formulations packaged in a BFS container.
  • the BFS container serves as a drug delivery device for administration (e.g., self administration) of the formulation, e.g., topical administration to the eye, e.g., as an eye drop.
  • an opaque package e.g., an aluminum foil pouch, over the BFS container protects the packaged formulation from light-induced degradation.
  • sealing the BFS container in an airtight package e.g., an aluminum foil pouch, typically with an inert gas (e.g., nitrogen) overlay, protects the packaged formulation against oxidation.
  • an airtight package e.g., an aluminum foil pouch
  • an inert gas e.g., nitrogen
  • One embodiment is directed to a method of providing a stable formulation comprising a chimeric cytokine protein described herein (e.g., an IL-i /IL-lRa chimeric cytokine protein, e.g., P05) in a blow-fill-seal (BFS) container.
  • a chimeric cytokine protein described herein e.g., an IL-i /IL-lRa chimeric cytokine protein, e.g., P05
  • BFS blow-fill-seal
  • the chimeric cytokine protein comprises a chimeric interleukin-1 (IL-1) family cytokine domain wherein at least a first segment of the domain is at least 20 amino acids in length and has at least 80% amino acid identity to a corresponding segment of a first IL-1 family cytokine, and at least a second segment of the domain is at least 20 amino acids in length and has at least 80% amino acid identity to a corresponding segment of a second IL-1 family cytokine.
  • the first IL-1 family cytokine is an IL-1 receptor agonist and the second IL-1 family cytokine is an IL-1 receptor antagonist.
  • the first and second IL-1 family cytokines are selected from the group consisting of IL- ⁇ , IL-la, and IL-lRa.
  • the chimeric cytokine protein comprises or consists of a sequence at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), or P05 (SEQ ID NO:5).
  • the chimeric cytokine protein is selected from the group consisting of P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), and P05 (SEQ ID NO:5). In embodiments, the chimeric cytokine protein is selected from the group consisting of P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), and P05 (SEQ ID NO:5).
  • the chimeric cytokine protein comprises or consists of P05.
  • the method may comprise dispensing the formulation into the BFS container with a BFS apparatus that includes a BFS dispenser that dispenses the formulation into the BFS container and a BFS transfer system configured to transfer the formulation to the BFS dispenser, the BFS transfer system having one or more cooling features configured to maintain the formulation at a low temperature prior to dispensing the formulation into the BFS container.
  • a BFS apparatus that includes a BFS dispenser that dispenses the formulation into the BFS container and a BFS transfer system configured to transfer the formulation to the BFS dispenser, the BFS transfer system having one or more cooling features configured to maintain the formulation at a low temperature prior to dispensing the formulation into the BFS container.
  • the cooling features comprise or consist of one or more lines in the BFS transfer system that are insulated and/or cooled.
  • the cooling features comprise a heat exchanger.
  • the heat exchanger may be configured to circulate refrigerant around one or more lines in the BFS transfer system.
  • Embodiments of the method may further comprise monitoring the temperature of the formulation in the BFS transfer system, for example, using a thermocouple, at a position adjacent to the dispenser.
  • the temperature of the formulation at the position of the thermocouple is less than 15°C.
  • the temperature of the formulation at the position of the thermocouple is 2-15°C.
  • the temperature of the formulation at the position of the thermocouple is less than 10°C.
  • the temperature of the formulation at the position of the thermocouple is 2-10°C.
  • the temperature of the formulation at the position of the thermocouple is about 8°C.
  • the temperature of the formulation at the position of the thermocouple is 8°C.
  • Embodiments of the method may further comprise maintaining the formulation at a temperature of 2-8°C in a holding tank within the BFS apparatus, and wherein the BFS transfer system is configured to transfer the formulation from the holding tank to the dispenser.
  • the formulation is an aqueous formulation comprising 1 to 50 mg/ml (e.g., 20 mg/ml, 10 mg/ml, or 5 mg/ml) of a chimeric cytokine as described herein, e.g., an IL-i /IL-lRa chimeric cytokine protein, e.g., P05.
  • the formulation is an aqueous formulation comprising a citrate buffer, e.g., sodium citrate.
  • the formulation is an aqueous formulation comprising a sugar.
  • the sugar is sucrose.
  • the formulation comprises sucrose, citrate, and a poloxamer, e.g., poloxamer 188.
  • the formulation comprises 8-12 mM sodium citrate, 4% to 6% sorbitol (w/v), 0.08% to 0.12% poloxamer 188 (w/v), and optionally sodium carboxymethyl cellulose (e.g., 0.1 to 1% (w/v)), wherein the formulation has a pH of 5.5 to 7.5.
  • the formulation consists of 1 to 50 mg/ml (e.g., 1 to 20 mg/ml, e.g. 20 mg/ml, 10 mg/ml, or 5 mg/ml) of a chimeric cytokine protein described herein, e.g., an IL-i /IL-lRa chimeric cytokine protein, e.g., P05; 8-12 mM sodium citrate; 4% to 6% sorbitol (w/v); and 0.08% to 0.12% poloxamer 188 (w/v); wherein the formulation has a pH of 5.5 to 7.5.
  • a chimeric cytokine protein described herein e.g., an IL-i /IL-lRa chimeric cytokine protein, e.g., P05; 8-12 mM sodium citrate; 4% to 6% sorbitol (w/v); and 0.08% to 0.12% poloxamer 188 (w/v); wherein the
  • the formulation does not comprise sodium carboxymethyl cellulose.
  • the formulation may comprise 1-20 mg/ml of a chimeric cytokine protein, e.g., P05.
  • the formulation consists of 10 mM sodium citrate, 5% sorbitol, 0.1% poloxamer 188, and 1-20 mg/ml P05.
  • Another embodiment is directed to a stable aqueous formulation packaged in a BFS container, the formulation comprising 1-20 mg/ml of a chimeric cytokine as described herein, e.g., an IL-i /IL- lRa chimeric cytokine protein, e.g., P05, 8-12 mM sodium citrate, 4% to 6% sorbitol (w/v), and 0.08% to 0.12% poloxamer 188 (w/v), wherein the formulation has a pH of 5.5 to 7.5. In embodiments, the formulation has a pH of 5.5 to 6.5.
  • a chimeric cytokine as described herein, e.g., an IL-i /IL- lRa chimeric cytokine protein, e.g., P05, 8-12 mM sodium citrate, 4% to 6% sorbitol (w/v), and 0.08% to 0.12% poloxamer 188 (w/v)
  • the BFS container is in a package, e.g., an aluminum foil pouch, optionally with an inert gas (e.g., nitrogen) overlay.
  • the package protects the formulation from light-induced degradation.
  • the package and/or inert gas overlay protect the formulation from oxidation. Oxidation of the formulation may be assessed using RP-HPLC, for example.
  • the formulation is stable after storage in the BFS container (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17 or 18 months) at room temperature, e.g., at 25°C.
  • the stability of the formulation is assessed after storage (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17 or 18 months) at 2-8 °C and 60% relative humidity.
  • the stability of the formulation can be assessed using one or more measures described herein, e.g., appearance, pH, content by absorbance at 280 nm (A280 or A 280 ), SDS-PAGE reduced, SDS-PAGE nonreduced, SE-HPLC, RP- HPLC, CIEX-HPLC, and potency.
  • the formulation satisfies a specification disclosed herein after storage in a BFS container.
  • the formulation has >90 (a/a) monomer as assessed using SE-HPLC.
  • the abbreviation "(a/a)" means that the percent is by peak area, relative to the total area.
  • the formulation has >80% (a/a) main peak as assessed using RP-HPLC.
  • the formulation has > 85% (a/a) main peak as assessed using CIEX-HPLC. In embodiments, the formulation has ⁇ 10 (a/a) des-Ala form as assessed using CIEX-HPLC. In embodiments, the formulation has an IC 50 of 60-140% of the reference standard IC 50 . In embodiments, the concentration of the formulation as assessed by A280 is within 10% of its target concentration. In embodiments, the content of a formulation with a target concentration of 5 mg/mL is 5 ⁇ 0.5 mg/mL as assessed by A280. In embodiments, the pH of the formulation after storage is 5.7 to 6.3.
  • the formulation satisfies a specification disclosed herein after storage in a BFS container, or has even better stability than needed to meet the specification.
  • the formulation has > 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (a/a) monomer as assessed using SE- HPLC.
  • the formulation has >80, 81, 82, 83, 84, 85, 86, 87, 87, 89, or 90% (a/a) main peak as assessed using RP-HPLC.
  • the formulation has > 85% (a/a) main peak as assessed using CIEX-HPLC.
  • the formulation has ⁇ 10, 9, 8, 7, 6, or 5% (a/a) des-Ala form as assessed using CIEX-HPLC. In embodiments, the formulation has ⁇ 5, 4, 3, 2, or 1% methionated species as assessed using CIEX-HPLC.
  • the formulation is stable as indicated by the presence of less than or equal to 50 particles per ml for particles >10 ⁇ , and/or less than or equal to 5 particles per ml for particles >25 ⁇ as assessed using a light obscuration particle count test.
  • the formulation is stable as indicated by the presence of > 90% of the monomeric form of the protein relative to aggregated form after vortexing the protein solution for 4 hours at room temperature, e.g., at 25°C.
  • the percentage in the formulation of the monomeric form of the protein relative to aggregated form may be assessed using SEC-HPLC, for example.
  • the formulation is stable after storage in the BFS container for at least 12, 13, 14, 15, 16, 17, 18, or 24 months at 2-8 °C and 60% relative humidity.
  • the formulation is stable after storage in the BFS container for at least 6 months at 2-8 °C and 60% relative humidity. In another example, the formulation is stable after storage in the BFS container for at least 6 months under ambient conditions, e.g., at room temperature, e.g., at 25°C. In one example, the formulation is stable after storage in the BFS container for at least 5 months at 2-8 °C and 60% relative humidity. In another example, the formulation is stable after storage in the BFS container for at least 5 months under ambient conditions, e.g., at room temperature, e.g., at 25°C.
  • the formulation is stable after storage in the BFS container for at least 4 months at 2-8 °C and 60% relative humidity. In another example, the formulation is stable after storage in the BFS container for at least 4 months under ambient conditions, e.g., at room temperature, e.g., at 25°C.
  • the formulation is stable after storage in the BFS container for at least 3 months at 2-8 °C and 60% relative humidity. In another example, the formulation is stable after storage in the BFS container for at least 3 months under ambient conditions, e.g., at room temperature, e.g., at 25°C.
  • the formulation is stable after storage in the BFS container for at least 2 months at 2-8 °C and 60% relative humidity. According to another example, the formulation is stable after storage in the BFS container for at least 2 months under ambient conditions, e.g., at room temperature, e.g., at 25°C.
  • the formulation is stable after storage in the BFS container for at least 1 month at 2-8 °C and 60% relative humidity. In another example, the formulation is stable after storage in the BFS container for at least 1 month under ambient conditions, e.g., at room temperature, e.g., at 25°C.
  • the formulation is stable as indicated by the presence of less than or equal to 50 particles per ml for particles >10 ⁇ and less than or equal to 5 particles per ml for particles >25 ⁇ , e.g., as assessed using a light obscuration particle count test.
  • the formulation is stable as indicated by the presence of > 90% of the monomeric form of the protein relative to aggregated form as assessed using SEC-HPLC.
  • the formulation is stable as indicated by conformity of the main band to reference standard in a reduced SDS-PAGE.
  • the formulation is stable as indicated by conformity of the main band to reference standard in a nonreduced SDS-PAGE.
  • the formulation is stable as indicated by a main peak of greater than or equal to 85% when the formulation is assessed using weak cation exchange HPLC or WCEX-HPLC.
  • the formulation comprises P05 and is stable as indicated by the presence of less than 10% of the des-Ala form of P05 as assessed using weak cation exchange HPLC or WCEX-HPLC.
  • the formulation comprises P05 and is stable as indicated by the presence of ⁇ 5, 4, 3, 2, or 1% methionated species as assessed using CIEX-HPLC.
  • a kit comprising a formulation packaged in a BFS container as described herein, and optionally, instructions for use.
  • FIG. 1 is a block diagram of an example of a BFS packaging system.
  • FIG. 2 is flow diagram of one example of a method of producing a formulation-filled BFS container.
  • FIG. 3A is a graph showing the dynamic light scattering (DLS) results for P05 in a phosphate formulation (P05 at 20 mg/ml, 10 mM phosphate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.5).
  • DLS dynamic light scattering
  • FIG. 3B is a graph showing DLS results for P05 in a citrate formulation (P05 at 20 mg/ml, 10 mM citrate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.0).
  • FIG. 4A is a graph showing the numbers of particles per mL as assessed over time using a light obscuration particle count test for particles >25 ⁇ and for particles >10 ⁇ .
  • the graph also shows the USP ⁇ 789> specifications for particulate matter in ophthalmic solutions, which are ⁇ 50 particles per mL for particles >10 ⁇ and ⁇ 5 particles per mL for particles >25 ⁇ .
  • FIG. 4B is a graph showing the numbers of particles per mL as assessed over time using a light obscuration particle count test for particles of 2 to 10 ⁇ .
  • BFS blow fill seal
  • aspects and embodiments are directed to methods of BFS processing to produce a stable formulation in a BFS container. Further aspects and embodiments are directed to a stable formulation in a BFS container.
  • the formulations are for ophthalmic delivery, e.g., for topical ophthalmic delivery.
  • formulation refers to a formulation containing one or more chimeric cytokine proteins as described herein.
  • the formulation may also include other components. Exemplary formulations and components are described in WO2014/160371 and in US2014/0308239.
  • An exemplary formulation described herein is aqueous and includes 10 mM sodium citrate, pH 6.0, 5% sorbitol (w/v) and 0.1% poloxamer (w/v).
  • the system includes a holding tank 110 that contains the formulation, a BFS filling system or dispenser 120, and a transfer system 130 that transfers the formulation from the holding tank 110 to the dispenser 120.
  • the holding tank 110 can be used to store the formulation for a short or extended time period to accommodate manufacturing scheduling.
  • the formulation may be prepared for the BFS process, and that this preparation can include adding a formulation buffer if the drug substance supplied for the formulation has a concentration different from the target concentration desired for the final product, and optionally filtering the formulation to reduce bioburden level.
  • the dispenser 120 is configured to fill a plurality of BFS containers 140 with the formulation in accord with conventional BFS filling techniques. After being filled, the BFS containers 140 are hermetically sealed in accord with conventional BFS processes and techniques.
  • the formulation may be maintained in the holding tank 110 at a temperature in a range of about 2-8° C.
  • the holding tank 110 may therefore be insulated.
  • the holding tank 110 may be actively cooled, for example, using circulating refrigerant supplied via a port 112. Methods of insulating and cooling are known in the art. In such examples, the formulation may be provided to the holding tank 110 already cool, or may be cooled in the holding tank from a slightly higher temperature.
  • the transfer system 130 may be insulated and/or actively cooled.
  • the transfer system 130 includes lines 132 (such as, tubes, pipes, or hoses, for example) through which the formulation is flowed from the holding tank 110 to the dispenser 120.
  • the transfer system optionally further includes a filter 134, a surge tank 136, and/or a heat exchanger 138.
  • the filter 134 may be a sterilizing filter, for example.
  • at least certain sections of the lines 132 that conduct the formulation to and from the various components of the transfer system 130 may be insulated or cooled.
  • the transfer system 130 may include a first section of cooled or insulated line 132a that conveys the formulation from the cooled and/or insulated holding tank 110 to the filter 134, and a second section of cooled/insulated line 132b that conveys the formulation from the heat exchanger 138 to the dispenser 120.
  • a third section of line 132c that conducts the formulation from the surge tank 136 to the heat exchanger 138 may also be insulated and/or cooled.
  • These cooled and/or insulated lines may include blue NPD hoses, white silicon wrapped hoses, or lines that are wrapped with insulated sheathing or foam.
  • the surge tank 136 may be used, for example, to control the volume and/or flow rate of formulation supplied to the dispenser 120.
  • the heat exchanger 138 may be used to re -cool the formulation prior to its reaching the dispenser in case any heating of the formulation occurred during the transfer from the holding tank 110 to the heat exchanger.
  • the formulation may heat up slightly during the time it is held in the surge tank. Accordingly, the heat exchanger 138 may be used to lower the temperature of the formulation.
  • the heat exchanger 138 may circulate refrigerant around the lines 132a, 132b, and 132c of the transfer system 130 to cool the lines and therefore the formulation flowed through the lines.
  • a thermocouple 150 may be coupled to the line section 132b, just prior to the point at which the formulation enters the dispenser, such that the temperature of the formulation may be measured just before the formulation is dispensed into the BFS containers 140. This information may be used to adjust the cooling aspects of the system to control the temperature of the formulation. For example, if the measured temperature of the formulation is above the desired temperature range, the cooling settings of the holding tank 110 and/or heat exchanger 138 may be adjusted to further cool the formulation.
  • the temperature of the formulation can be controlled prior to the filling step of the BFS process.
  • a relatively low temperature e.g., at a temperature of 15°C or less, at a temperature of 10°C or less, or at a temperature of 8°C or less
  • the effects of contacting the warm BFS containers can be minimized, thereby allowing production of stable packaged formulations.
  • the formulation includes a chimeric cytokine protein described hereinin a concentration range of 0.5 mg/ml to 30 mg/ml, for example, from 1 mg/ml to 30 mg/ml, 1 mg/ml to 20 mg/ml, 1 mg/ml to 5 mg/ml, or from 5 mg/ml to 20 mg/ml.
  • concentration range of 0.5 mg/ml to 30 mg/ml, for example, from 1 mg/ml to 30 mg/ml, 1 mg/ml to 20 mg/ml, 1 mg/ml to 5 mg/ml, or from 5 mg/ml to 20 mg/ml.
  • the formulation may include a buffer that also provides increased stability and resistance to temperature effects, as demonstrated in the examples provided below.
  • a citrate buffer sodium citrate
  • the formulation includes sodium citrate.
  • the temperature of onset of unfolding of the chimeric cytokine protein in the formulation is at least 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48°C.
  • a chimeric cytokine protein as described herein, e.g., P05 is formulated at concentrations of 5 mg/ml to 20 mg/ml (e.g., at a concentration of 1 mg/ml, 5 mg/ml, or 20 mg/ml) in 5- 15 mM (e.g., 10 mM) sodium citrate, pH 5.5-6.5 (e.g., pH 6.0) containing 2.5-7.5% (e.g., 5%) w/v sorbitol and 0.05-0.15% (e.g., 0.1%) w/v poloxamer, e.g., poloxamer 188 (also referred to as, for example, Lutrol® F-68 or Kolliphor® P 188).
  • the chimeric cytokine protein is selected from one or more of P01, P02, P03, P04, P05, P06, and P07. In embodiments, the chimeric cytokine is P05.
  • the components of a formulation described herein are present in amounts that may vary around the values provided herein by up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In embodiments, the components of a formulation are present in amounts that vary around the values provided herein by 10%.
  • the formulation comprises 9.5-10.5 mM, 9-11 mM, 8.5-11.5 mM, 8-12 mM, 7.5-12.5 mM, 7-13 mM, 6-14 mM, or 5-15 mM sodium citrate.
  • the formulation comprises 4.75-5.25%, 4.5-5.5%, 4.25-5.75%, 4-6%, 3.75- 6.25%, 3.5-6.5%, 3-7%, or 2.5-7.5% w/v sorbitol.
  • the formulation comprises 0.095-0.105%, 0.09-0.11%, 0.085-0.115%, 0.08- 0.12%, 0.075-0.125%, 0.07-0.13%, 0.06-0.14%, or 0.05-0.15% w/v poloxamer 188.
  • the concentration of the chimeric cytokine protein (e.g., P05) in the formulation is 1-50 mg/ml, 1-25 mg/ml, or 1-20 mg/ml. In embodiments, the concentration of the chimeric cytokine protein is 4.75-5.25 mg/ml, 4.5-5.5 mg/ml, 4.25-5.75 mg/ml, 4-6 mg/ml, 3.75-6.25 mg/ml, 3.5-6.5 mg/ml, 3-7 mg/ml, or 2.5-7.5 mg/ml. In embodiments, the pH of the formulation is 5.5 to 7.5, or 5.5 to 6.5.
  • the formulation comprises 8-12 mM sodium citrate, 4-6% w/v sorbitol, 0.08- 0.12% w/v poloxamer 188, and 4-6 mg/ml of a chimeric cytokine protein, e.g., P05.
  • the pH of the formulation is 5.5 to 7.5. In embodiments, the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7.
  • the formulation comprises 9-11 mM sodium citrate, 4.5-5.5% w/v sorbitol, 0.09-0.11% w/v poloxamer 188, and 4.5-5.5 mg/ml of a chimeric cytokine protein, e.g., P05.
  • the pH of the formulation is 5.5 to 7.5. In embodiments, the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7.
  • the formulation comprises 10 mM sodium citrate, pH 6.0 containing 5% w/v sorbitol and 0.1% w/v poloxamer, e.g., poloxamer 188.
  • the amounts of the components of the formulation may vary around the values provided herein by up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%.
  • the pH is 5.5 to 7.5.
  • the pH is 5.5 to 6.5.
  • the pH is 6 to 7.
  • Topical ophthalmic drugs are generally self-administered by patients. Because the patient may be storing a drug for a relatively long period of time, the formulation may be subjected to higher temperatures and greater levels of agitation stress than a formulation that is typically stored only by a physician or pharmacist prior to administration. As is known in the art, proteins are more sensitive to agitation and temperature than small molecules. Agitation stress can lead to precipitation and heat stress can lead to precipitation and to chemical degradation. In addition, during loading of a compound into a BFS delivery device, there can be exposure to heat stress. The formulations used herein provide excellent stability when exposed to agitation stress and heat.
  • a formulation referred to herein is stable.
  • the formulation exhibits stability immediately following dispensation into a BFS container and/or after storage in a BFS container. Exemplary conditions and measurements that can be used to assess stability are discussed in further detail below.
  • the formulation is stable in a BFS container at about 25°C to about 40°C, for example, about 27°C, about 28°C, about 29°C , about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C for a period of at least two days; three days; five days; one week; ten days, two weeks, three weeks, four weeks, five weeks, six weeks, eight weeks, 16 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, or more.
  • Stability can be assessed, e.g., after storage for at least 2, 4, 6, 8, 12, 15, 16, 17, or 18 months, e.g., at 2-8°C, or after storage under ambient conditions, e.g., at room temperature (RT), e.g. at about 25°C for, e.g., at least 2 weeks, 1 month, 2 months, 3 months 5 months, 6 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months or 18 months.
  • the formulation is stable after storage at 2-8°C for at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months.
  • the formulation is stable after exposure to room temperature for at least 3, 4, 5, or 6 months.
  • the formulation is stable after storage, e.g., for at least 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, in a BFS container.
  • Stability can be assessed, e.g., based on methods and criteria described herein or known in the art. For example, stability can be assessed based on physical purity (e.g., lack of aggregation, e.g., as assessed using size exclusion HPLC, also referred to herein as size exclusion, SE HPLC, or SEC HPLC), chemical purity (e.g., as assessed using weak cation exchange HPLC , reverse phase HPLC, and/or SDS PAGE (e.g., reduced or nonreduced SDS PAGE)), and/or the levels of particulates (e.g., as assessed visually or by particle count using an HIAC liquid particle counter (Beckman Coulter, Brea, CA)).
  • physical purity e.g., lack of aggregation, e.g., as assessed using size exclusion HPLC, also referred to herein as size exclusion, SE HPLC, or SEC HPLC
  • chemical purity e.g., as assessed using weak cation exchange HPLC
  • Stability can also be assessed based on visual appearance.
  • the formulation is stable if it is a clear to slightly opalescent colorless solution essentially free from visible particulates.
  • stability is demonstrated based on compliance with guidelines for particulate matter in ophthalmic solutions, e.g., as set forth in USP ⁇ 789> (U.S. Pharmacopeia, Particulate Matter in Opthalmic Solutions).
  • the formulation has less than or equal to 50 particles per ml for particles >10 ⁇ and/or less than or equal to 5 particles per ml for particles >25 ⁇ , e.g., as assessed using a light obscuration particle count test (e.g., a light obscuration particle count test as described in USP ⁇ 788>).
  • a light obscuration particle count test e.g., a light obscuration particle count test as described in USP ⁇ 788>.
  • the formulation has less than or equal to 50 particles per ml for particles >10 ⁇ , less than or equal to 5 particles per ml for particles >25 ⁇ , and/or less than or equal to 2 particles per ml for particles >50 ⁇ , e.g., as assessed using a microscopic particle count test (e.g., a microscopic particle count test as described in USP ⁇ 788>).
  • a microscopic particle count test e.g., a microscopic particle count test as described in USP ⁇ 788>.
  • stability is demonstrated based on compliance with guidelines for particulate matter in injections, e.g., as set forth in USP ⁇ 788> (U.S. Pharmacopeia, Particulate Matter in
  • the formulation has less than or equal to 6000 particles per container (for containers with a volume of 100 ml or less) for particles >10 ⁇ , and/or less than or equal to 600 particles per container (for containers with a volume of 100 ml or less) for particles >25 um, e.g., as assessed using a light obscuration particle count test (e.g., a light obscuration particle count test as described in USP ⁇ 788>).
  • a light obscuration particle count test e.g., a light obscuration particle count test as described in USP ⁇ 788>.
  • the formulation has less than or equal to 3000 particles per 5 ml for particles >10 ⁇ and/or less than or equal to 300 particles per 5 ml for particles >25 um, e.g., as assessed using a microscopic particle count test (e.g., a microscopic particle count test as described in USP ⁇ 788>).
  • a microscopic particle count test e.g., a microscopic particle count test as described in USP ⁇ 788>.
  • the protein in a formulation is resistant to agitation stress as demonstrated, e.g., by lack of aggregation (lack of aggregation may be demonstrated, e.g., if the formulation contains > 90%, > 91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99% of the monomeric form of the protein relative to aggregated form) after vortexing the protein solution, e.g., for 1-8 hours at room temperature (RT), e.g., for 4 hours at RT.
  • Aggregation can be assessed, e.g., using methods described herein or methods known in the art. For example, aggregation can be assessed using ultracentrifugation, size-exclusion chromatography, gel electrophoresis, dynamic light scattering, and/or turbidity measurements.
  • stability is assayed by physical or chemical methods known in the art. For example, physical purity or lack of aggregation can be determined using size exclusion HPLC or other methods that determine the relative amount of monomeric polypeptide in a formulation.
  • a formulation with acceptable stability contains > 90% of the monomeric form of therapeutic protein (e.g., the chimeric cytokine, e.g., P05) relative to aggregated forms of the protein.
  • the formulation contains > 90% (e.g., > 91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) of the monomeric form of the therapeutic protein (e.g., the chimeric cytokine, e.g., P05), relative to aggregated forms of the protein.
  • the monomeric form of the therapeutic protein e.g., the chimeric cytokine, e.g., P05
  • Chemical purity can be determined, for example, using weak cation exchange HPLC or reverse phase HPLC.
  • a formulation with acceptable stability contains > 80% of the native molecule, relative to chemically modified forms of the molecule, e.g., as assessed using weak cation exchange HPLC.
  • the formulation contains > 80% (e.g., > 85%, > 87%, > 90%, or >95%) of the native molecule, relative to chemically modified forms of the molecule (e.g., oxidized or acetylated forms).
  • Particulates may be identified visually.
  • the formulation is one that is essentially free of particulates that can be identified visually.
  • Biologic treatments can be problematic to administer because they can have a relatively short shelf life or require special storage conditions that can create obstacles for storage, transport, and patient use as well as assuring a sufficient supply of the biologic.
  • An advantage of certain formulations provided herein is that the formulations are surprisingly stable not only under conditions of
  • the formulations are typically in a liquid form that is stable at room temperature (RT) (e.g., at 25°C) for a period of at least three days, five days, one week, ten days, two weeks, three weeks, six weeks, eight weeks, 16 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, twelve months, or more.
  • a month is determined on date to date basis, e.g., from the first of the month to the first of the second month.
  • the formulations are stable at about 25°C to about 40°C, for example, about 27°C, about 28°C, about 29°C , about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C for a period of at least two days; three days; five days; one week; ten days, two weeks, three weeks, four weeks, five weeks, six weeks, eight weeks, 16 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, or more.
  • a formulation is stable for one month at 25°C and 1 week at 40°C when the protein component of the formulation, e.g., P05, is at a concentration of 20 mg/ml.
  • the formulation is stable at 25°C for at least three months for a formulation comprising protein, e.g., P05, at a concentration of 1-20 mg/ml, e.g., about 1 mg/ml, 5 mg/ml, or 10 mg/ml. In some embodiments, this formulation is stable for at least five months.
  • a formulation comprising 4.5-5.5 mg/ml P05, 9-11 mM sodium citrate; 4.5- 5.5% w/v sorbitol, and 0.09-0.11% w/v poloxamer 188 is stable for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months at 2°C to 8°C and/or at room temperature, e.g., at 25°C.
  • a formulation consisting of 10 mM Na citrate, pH 6.0, 5% sorbitol, 0.1% poloxamer, and 5 mg/ml or 20 mg/ml P05 is stable for at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months at 2°C to 8°C and/or is stable at room temperature, e.g., at 25°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months.
  • a formulation consisting of 10 mM Na citrate, pH 6.0, 5% sorbitol, 0.1% poloxamer, and 5 mg/ml or 20 mg/ml P05 is stable for at least five months at 2°C to 8°C and/or at room temperature, e.g., at 25°C for at least 5 months.
  • a first step 210 includes preparing and cooling the formulation to be packaged using the BFS process. As discussed above, this step 210 may include providing the bulk drug substance (e.g., P05), adding desired formulation components, e.g., a formulation buffer (such as a citrate buffer, as discussed above), to produce the desired formulation, and cooling the formulation to a desired temperature range either in the holding tank 110 or before placing the formulation in the holding tank.
  • Step 220 includes transferring the formulation to the BFS dispenser via the BFS transfer system 130.
  • the transfer system 130 may include features such as cooled/insulated lines 132a, 132b, and 132c, and/or active cooling mechanisms such as the heat exchanger 138. These cooling features may be configured to cool and/or maintain the formulation at the desired temperature range.
  • the BFS containers may then be filled with the formulation (step 230), and hermetically sealed (step 240) using conventional BFS processes.
  • the use of the formulations discussed herein, in combination with the transfer system 130 that includes cooling features allows stable packaged formulations to be provided using the BFS process, contrary to expectations based on conventional wisdom in the art.
  • the formulations referred to herein are typically aqueous formulations.
  • the formulations referred to herein include a chimeric cytokine protein.
  • a chimeric cytokine protein refers to a protein that comprises an IL-1 family chimeric cytokine domain as described in WO 2012/016203 or WO 2012/103240, the entire contents of which are hereby incorporated by reference.
  • the chimeric cytokine protein comprises a chimeric interleukin-1 (IL-1) family cytokine domain wherein at least a first segment of the domain is at least 20 amino acids in length and has at least 80% amino acid identity to a corresponding segment of a first IL- 1 family cytokine, and at least a second segment of the domain is at least 20 amino acids in length and has at least 80% amino acid identity to a corresponding segment of a second IL-1 family cytokine.
  • IL-1 interleukin-1
  • the first IL-1 family cytokine is an IL-1 receptor agonist and the second IL-1 family cytokine is an IL-1 receptor antagonist.
  • the first and second IL-1 family cytokines are selected from the group consisting of IL- ⁇ , IL-la, and IL-lRa.
  • the first and second IL-1 family cytokines are IL- ⁇ and IL-lRa, respectively.
  • the protein consists of a single polypeptide chain that is 150-160 amino acids in length.
  • the chimeric cytokine protein can act as an IL-1 inhibitor.
  • the chimeric cytokine domain binds to IL-1 receptor I (IL-1RI) (e.g., with a Kd of less than 5 nM).
  • IL-1RI IL-1 receptor I
  • the chimeric cytokine domain inhibits IL-1RI activity .
  • the chimeric cytokine domain inhibits signaling by IL- ⁇ at a concentration of 0.1 ng/ml with an IC50 of less than 50 nM.
  • the protein inhibits IL-i -induced IL-6 expression in MG-63 cells.
  • the chimeric cytokine domain is not naturally occurring. In embodiments, the chimeric cytokine domain is less than 80% identical to human IL-lRa (SEQ ID NO: 18), human IL- ⁇ (SEQ ID NO: 16 and/or human IL-la (SEQ ID NO: 17).
  • IL-la human
  • IL-lRa human
  • IL- ⁇ IL- ⁇
  • IL-la IL-la
  • IL-lRa IL-lRa
  • the cytokine domain comprises at least two discontinuous segments of length at least 20 amino acids that are at least 90% identical to respective corresponding segments from human IL- ⁇ .
  • the two discontinuous segments are identical to respective corresponding segments from human IL-1 ⁇ .
  • the two discontinuous segments are between 25-40 amino acids in length.
  • the amino acids not in the two discontinuous segments are at least 90% identical to human IL-lRa.
  • the chimeric cytokine protein is an IL-i /IL-lRa chimeric cytokine protein, e.g., P05.
  • the formulations referred to herein include a protein or polypeptide that comprises or consists of P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), P05 (SEQ ID NO:5), P06 (SEQ ID NO:6), P07 (SEQ ID NO:7), P08 (SEQ ID NO:8), P09 (SEQ ID NO:9), P10 (SEQ ID NO: 10), Pl l SEQ ID NO: 11), P12 SEQ ID NO: 12), P13 SEQ ID NO: 13), P14 (SEQ ID NO: 14), or SEQ ID NO: 15. These sequences are provided below.
  • the formulations referred to herein include an IL-i /IL-lRa chimeric cytokine protein that comprises or consists of P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), P05 (SEQ ID NO:5), P06 (SEQ ID NO:6), or P07 (SEQ ID NO:7).
  • the formulations referred to herein include a protein that comprises a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), P05 (SEQ ID NO:5), P06 (SEQ ID NO:6), P07 (SEQ ID NO:7), P08 (SEQ ID NO:8), P09 (SEQ ID NO:9), P10 (SEQ ID NO: 10), Pl l (SEQ ID NO: 11), P12 SEQ ID NO: 12), P13 SEQ ID NO: 13), P14 (SEQ ID NO: 14), or SEQ ID NO: 15.
  • the chimeric cytokine protein differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids from P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), P05 (SEQ ID NO:5), P06 (SEQ ID NO:6), P07 (SEQ ID NO:7), P08 (SEQ ID NO:8), P09 (SEQ ID NO:9), P10 (SEQ ID NO: 10), Pl l (SEQ ID NO: 11), P12 SEQ ID NO: 12), P13 SEQ ID NO: 13), P14 (SEQ ID NO: 14), or SEQ ID NO: 15.
  • the formulations referred to herein include a protein that comprises a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), or P05 (SEQ ID NO:5).
  • the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P01 (SEQ ID NO: l), P02 (SEQ ID NO:2), P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), or P05 (SEQ ID NO:5).
  • the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P01 (SEQ ID NO: l).
  • the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P02 (SEQ ID NO:2).
  • the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P03 (SEQ ID NO:3).
  • the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P04 (SEQ ID NO:4). In embodiments, the formulations referred to herein include a protein that comprises a sequence at least 95% identical to P05 (SEQ ID NO:5).
  • the chimeric cytokine protein is more thermostable than human IL-lRa and human IL- ⁇ .
  • chimeric cytokine protein includes an IL-i /IL-lRa chimeric cytokine domain that has greater thermostability than IL- ⁇ and IL-lRa.
  • the formulation includes a chimeric cytokine protein having a chimeric IL-i /IL-lRa cytokine domain that includes one or more structural features associated with thermal stability, e.g., a unique salt bridge or hydrogen bond as described in Example 10 of WO 2012/016203.
  • the chimeric cytokine domain comprises one or more (e.g., 1, 2, or all 3) of (i) E44 from human IL-lRa and K65 from human IL- ⁇ ; (ii) R14 from human IL-lRa and Q149 from human IL- ⁇ ; and (iii) S152 from human IL- ⁇ and K45 from human IL-lRa.
  • the IL-i /IL-lRa chimeric cytokine protein comprises or consists of a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), or P05 (SEQ ID NO:5).
  • the chimeric cytokine protein comprises or consists of a sequence identical to P03 (SEQ ID NO:3), P04 (SEQ ID NO:4), or P05 (SEQ ID NO:5).
  • the chimeric cytokine domain exhibits one, two, three, four, or all of the following thermal properties (i) has a melting temperature (T m ) that is at least 2°C greater than human IL- ⁇ and human IL-lRa, (ii) has a T m that is at least 58°C (iii) has a T m that is greater than the T m of human IL-lRa and human IL- ⁇ , (iv) has a temperature of onset of unfolding that is greater than the T m of human IL-lRa and human IL- ⁇ , and (v) has a temperature of onset of unfolding that is at least 50°C, e.g., as assessed using differential scanning fluorimetry at a concentration of 0.5 mg/mL in PBS at pH 7.4, e.g., as described in Example 7 of WO 2012/016203.
  • the chimeric cytokine protein comprises or consists of P05 (SEQ ID NO:5).
  • a formulation described herein is for topical, e.g., topical ophthalmic administration.
  • the formulation is for administration as an eye drop.
  • the formulation is for administration by the subject.
  • a method of treatment described herein comprises self-administration of the formulation by the subject.
  • the BFS container serves as a drug delivery device for administration (e.g., self administration) of the formulation, e.g., topical administration to the eye, e.g., as an eye drop.
  • the protein is effective in modulating an immune or inflammatory response.
  • the formulation is for modulation of an immune or inflammatory response.
  • a method of modulating an immune or inflammatory response in a subject comprising administering to the subject a formulation described herein.
  • the protein is effective in treating an ocular disease.
  • the formulation is for treatment of an ocular disease.
  • a method of treating an ocular disease in a subject comprising administering to the subject a formulation described herein.
  • the ocular disease is dry eye disease or allergic conjunctivitis.
  • the protein is effective in treating allergic rhinitis.
  • formulation is for treatment of allergic rhinitis. Also provided herein is a method of treating an allergic rhinitis in a subject comprising administering to the subject a formulation described herein.
  • a method of treatment e.g., of an eye disease in a subject (e.g., dry eye disease or dry eye disorder), the method comprising administering (e.g., topically administering) to the subject a formulation described herein.
  • administering e.g., topically administering
  • the subject self- administers the formulation, e.g., by topically applying the formulation, e.g., to the eye, e.g., as an eye drop.
  • kits comprising a formulation in a BFS container as described herein and optionally instructions for use.
  • the container serves as a delivery device for administration (e.g., self administration) of the formulation, e.g., for topical administration, e.g., for topical application to the eye, e.g., as an eye drop.
  • Table 1 Exemplary IL-i /IL-lRa chimeric cytokine prot
  • Additional exemplary chimeric IL-1 family proteins also include the following:
  • the polypeptide below is a chimeric cytokine domain that includes at least two segments from IL-1 a and at least two segments from IL-lRa.
  • Dynamic light scattering or DLS measures the diffusion of an analyte (e.g., P05) in a well plate by focusing laser light on the sample, and monitoring the rate of fluctuation of the scattered light as measured by a fast photon counter.
  • a mathematical technique known as a correlation function, is used to quantify the rate of fluctuation to determine the diffusion coefficient.
  • the diffusion coefficient is used to obtain radius of hydration (Rh) by the Stokes-Einstein equation.
  • the radius of P05 was measured as a function of increasing temperature in a DLS plate reader (Wyatt DynaProTM, Wyatt Technologies, Santa Barbara, CA). The acquisition time was 5 seconds and 5 scans were performed for each measurement. The ramp rate was 0.17° CI min. As the protein unfolded, the radius increased. The temperature at which the radius increased is referred as T on (temperature of onset for unfolding).
  • a formulation comprises a citrate buffer.
  • Example 2 Stability of Formulations in Blow Fill Seal Containers
  • BFS blow fill seal
  • P05 active pharmaceutical
  • Bulk drug substance was formulated into an aqueous solution containing 10 mM sodium citrate, 5% w/v sorbitol, 0.1 % w/v poloxamer 188, at pH 6.0 for blow fill processing.
  • the target concentration for P05 was 5.0 mg/mL.
  • the number of units filled was approximately 1000 containers with a target fill volume of 0.32 mL.
  • the process stream was maintained at approximately 2-8 °C prior to entering the blow fill equipment, by insulating and cooling the transfer system using methods described herein, including insulation of the lines between the holding tank and dispenser, and use of a heat exchanger between the surge tank and dispenser.
  • a portion of the containers were pouched in foil packages with a nitrogen overlay.
  • An initial characterization was performed following the packaging into BFS containers and further stability evaluations were conducted following storage at two temperatures (2 to 8 °C and 25 °C), with or without pouching.
  • the initial characterization analysis included: concentration by A 280 , SDS-PAGE, SEC-HPLC, wCEX-HPLC, RP-HPLC, osmolality and particle analysis by light obscuration.
  • concentration by A 280 SDS-PAGE
  • SEC-HPLC SEC-HPLC
  • wCEX-HPLC wCEX-HPLC
  • RP-HPLC osmolality and particle analysis by light obscuration.
  • the stability of P05 was monitored monthly by SEC-HPLC, wCEX-HPLC and RP-HPLC.
  • a 280 evaluation and pH and osmolality assessments were performed at months 5, 12, and 18.
  • osmolality and concentration by A 280 were measured.
  • the average osmolality measurement was 317 mOsm, and the average concentration by A 280 was 5.0 mg/mL.
  • the P05 formulation in BFS containers was stored in an incubator at 25 °C with 60% relative humidity or at 2 to 8 °C.
  • samples were analyzed by SEC, RP-HPLC, and wCEX- HPLC.
  • SEC separates monomers from aggregates; formation of aggregates results in a decline in the main peak.
  • RP-HPLC provides separation based on hydrophobicity; oxidation of the protein (e.g., oxidation of methionines) leads to additional peaks.
  • wCEX-HPLC measures the formation of charge variants (deamidation leads to additional peaks).
  • concentration was measured by A 280 .
  • concentration was measured by A 280 .
  • pH and concentration by A 280 were also measured.
  • the SEC-HPLC results indicate that P05 stored in BFS containers did not form aggregates at room temperature (25°C) for at least 6 months, or at 2 to 8°C for at least 18 months. Pouching of the vials after nitrogen flushing did not affect the physical stability of the product at either temperature.
  • the wCEX-HPLC stability results for the P05 formulation are shown in Table 5 and Table 6 (% main peak and % deamidated peaks, respectively).
  • the RP-HPLC results indicate that the P05 formulation stored in BFS containers was stable at room temperature (25°C) for at least 6 months and at 2 to 8 °C for at least 18 months.
  • Vehicle is the aqueous solution including the same formulation components except that it does not contain P05.
  • thermocouple was placed immediately above the fill system (dispenser), so that the temperature of the formulation during the fill could be assessed.
  • the thermocouple indicated a temperature of 8°C during the fill
  • the thermocouple for the bulk solution in the compounding tank (holding tank) indicated a temperature of 3°C.
  • Tables 10-13 provide stability results for cGMP lots of formulated drug product, referred to herein as "Lot X" and “Lot Y”.
  • the data for 25°C stability for both lots are shown first (see Tables 10 and 11), followed by the data for storage at 2 to 8°C (see Tables 12 and 13).
  • Assessments included appearance, pH, content by A280, SDS-PAGE reduced, SDS-PAGE non-reduced, SE-HPLC, RP- HPLC, CIEX-HPLC, and potency.
  • SE-HPLC also referred to herein as SEC
  • SEC separates monomers from aggregates; formation of aggregates results in a decline in the main peak.
  • RP-HPLC provides separation based on hydrophobicity; oxidation of the protein (e.g., oxidation of methionines) results in a decline in the main peak.
  • CIEX-HPLC cation exchange HPLC
  • charge variants of the protein which result, e.g., from deamidation of asparagine to form aspartate or glutamine to form glutamate
  • formation of charge variants results in a decline in the main peak.
  • Table 10 P05 Phase 2/3 GMP Drug Product Lot X at 25 ⁇ 3 °C/60% Relative Humidity Stored
  • particulates particulates from visible particulates particulates

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Abstract

La présente invention concerne des formulations stables comprenant une protéine cytokine chimérique (par exemple, une protéine cytokine chimérique IL-1β/IL-1Ra , par exemple, P05) dans un récipient du type à soufflage-remplissage-fermeture (BFS), et des procédés de production de formulations de protéine cytokine chimérique dans des récipients BFS, de sorte que les formulations conservent leur stabilité.
PCT/US2015/020357 2014-03-13 2015-03-13 Procédés pour la production de formulations de protéines cytokine chimériques stables dans des récipients du type à soufflage/remplissage/fermeture WO2015138844A1 (fr)

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US11459376B2 (en) 2019-09-13 2022-10-04 Elektrofi, Inc. Compositions and methods for the delivery of therapeutic biologics for treatment of disease
US11654112B2 (en) 2016-11-22 2023-05-23 Elektrofi, Inc. Particles comprising a therapeutic or diagnostic agent and suspensions and methods of use thereof
US11717488B2 (en) 2019-01-31 2023-08-08 Elektrofi, Inc. Particle formation and morphology

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