TW201538178A - Methods for production of stable chimeric cytokine protein formulations in blow fill seal containers - Google Patents

Abstract

Provided herein are stable formulations comprising a chimeric cytokine protein (e.g., an IL-1[beta]/IL-1Ra chimeric cytokine protein, e.g., P05) in a blow-fill-seal (BFS) container, and methods of producing chimeric cytokine protein formulations in BFS containers such that the formulations retain stability.

Description

Stable chimeric cytokine protein formulation produced in a blow molded, sealed, sealed container Method Related application

This application claims the benefit of priority to U.S. Patent Application Serial No. 61/952, the entire disclosure of which is incorporated herein by reference.

Sequence table

The present application contains a sequence listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 10, 2015, with the name D2046-7058WO_SL.txt and a file size of 26,682 bits.

Field of invention

The present invention is directed to a method of producing a therapeutic biological agent.

Background of the invention

With regard to biopharmaceuticals delivered by certain routes, such as, for example, eye delivery (e.g., topical applications, such as eye drops), plastic ampoules provide several advantages, such as convenience, as well as ease of use and child safety. Of course However, the high temperatures of filling such ampoules are unfavorable conditions for biological agents, which are generally sensitive to thermal damage.

Plastic ampoules can be manufactured using blow molding fill seal (BFS) technology. The process of encapsulating a formulation using BFS technology generally involves plastic extrusion, molding of extruded plastic to form a BFS container (which may also be referred to as an ampoule, vial, or unit), filling the BFS container with the formulation. And sealing the filled container, and the steps are performed in sequence. See, for example, Liu, W. et al., 2011 Biopharm International, 24(7): 22-29, which is incorporated herein by reference in its entirety for all purposes.

The material used to form the BFS container is typically supplied as an asparaule of a thermoplastic resin, such as low density polyethylene or polypropylene. In terms of the extrusion step, the particles may be melted at a temperature above 160 °C. The plastic is then molded into the desired container shape, filled into the formulation, and sealed. The BFS container can be partially cooled during the molding step or prior to the filling step. For example, cooling water can be circulated around the mold, which can help reduce the temperature of the BFS container. However, this cooling process is limited by the relatively short contact time between the BFS container and the mold (typically only about 10 seconds), and the temperature at which the BFS container needs to be kept high enough to ensure that at the end of the filling process Proper sealing.

At least to some extent, due to the high temperature associated with the extrusion process, and the container must remain hot enough to form a seal during the sealing step, the formulation is exposed to potentially adverse effects during the packaging process The temperature at which the protein is stable. In addition, the plastic material used to form the BFS container is somewhat breathable. Thus, the stability of the formulation may be lost during long term storage (such as due to evaporation of water from the container and/or protein oxidation). Because of the thermally sensitive nature of therapeutic polypeptides, BFS processing is generally not suitable for the production of encapsulated therapeutic protein-containing formulations with acceptable stability. It is contemplated that because the formulation must be exposed to heat from the container during the filling process, the therapeutic protein may lose stability, such as shown in the form of microparticles, aggregates, and modified (such as oxidized) forms.

Summary of invention

Surprisingly, Applicants have discovered a chimeric cytokine formulation that has been successfully produced in a BFS container so that the formulation retains stability immediately after BFS processing and after extended storage. A method and apparatus for encapsulating a formulation comprising a chimeric cytokine protein is described herein using a process BFS.

Described herein is a method of BFS processing involving a temperature controlled transfer system, the formulation described herein is passed through the transfer system prior to dispensing into a BFS container, which allows for the production of a stable formulation in a blow molded filled sealed container. . Also described herein is a stable formulation packaged in a BFS container. In a specific example, the BFS container is suitable as a drug delivery device for administering the formulation, such as a self-administration, such as topical administration to the eye, such as, for example, an eye drop.

In a specific example, an opaque layer covering the BFS container A package, such as an aluminum foil pouch, protects the package's formulation from photoinduced degradation.

In a specific example, sealing the BFS container in a hermetic package, such as an aluminum foil pouch, typically accompanied by an inert gas (e.g., nitrogen) overlay, will protect the package formulation from oxidation.

One specific embodiment is directed to a method of providing a stable formulation in a blow molded fill seal (BFS) container comprising a chimeric cytokine protein as described herein (eg, an IL-1β/IL-1Ra inlay). A cytokine protein, such as P05). In a specific embodiment, the chimeric cytokine protein comprises a cytokine domain of a chimeric interleukin-1 (IL-1) family, wherein at least one first segment of the region has a length of at least 20 An amino acid and having at least 80% amino acid identity to a corresponding segment of the first IL-1 family cytokine, and at least one second segment of the region having a length of at least 20 amines The base acid and at least 80% amino acid identity to the corresponding segment of the second IL-1 family cytokine. In a specific example, 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. In a specific example, the first and second IL-1 family cytokines are selected from the group consisting of IL-1β, IL-1α, and IL-1Ra. In a specific example, the chimeric cytokine protein comprises the following or consists of: P01 (SEQ ID NO: 1), P02 (SEQ ID NO: 2), P03 (SEQ ID NO: 3), P04 (Sequence ID: 4), or P05 (Sequence ID: 5) has a sequence of at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity. . In a specific example, the chimeric type The cytokine protein is selected from the group consisting of P01 (sequence identification number: 1), P02 (sequence identification number: 2), P03 (sequence identification number: 3), and P04 (sequence identification number: 4). And P05 (sequence identification number: 5). In a specific example, 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) . In a specific example, the chimeric cytokine protein comprises P05 or consists of P05.

The method can include dispensing the formulation into the BFS container with a BFS device comprising a BFS dispenser to dispense the formulation into the BFS container, and a BFS delivery system configured to deliver the formulation to The BFS dispenser has one or more cooling features that are configured to maintain the formulation at a low temperature prior to dispensing the formulation into the BFS container.

In one example, the cooling component comprises or consists of one or more lines within the BFS delivery system that are insulated and/or cooled. In another example, the cooling component comprises a heat exchanger. The heat exchanger can be configured to circulate the refrigerant around one or more of the lines within the BFS delivery system.

Specific examples of the method can be further included at a location adjacent to the dispenser, for example using a thermocouple to monitor the temperature of the formulation within the BFS delivery system. In one example, the temperature of the formulation is less than 15 ° C at the location of the thermocouple. In another example, the temperature of the formulation is 2-15 ° C at the location of the thermocouple. In another example, the temperature of the formulation is less than 10 ° C at the location of the thermocouple. On another In the example, the temperature of the formulation is 2-10 ° C at the location of the thermocouple. In another example, the temperature of the formulation at the location of the thermocouple is about 8 °C. In another example, the temperature of the formulation at the location of the thermocouple is 8 °C.

A specific example of the method can further comprise maintaining the temperature of the formulation in the storage tank in the BFS unit at 2-8 ° C, and wherein the BFS delivery system is configured to transfer the formulation from the storage tank to the dispensing Inside the device.

Typically, the formulation is an aqueous formulation comprising a chimeric cytokine of 1-50 mg/ml (such as 20 mg/ml, 10 mg/ml, or 5 mg/ml) as described herein, for example, a IL-1β/IL-1Ra chimeric cytokine protein, such as P05. In a specific example, the formulation is an aqueous formulation comprising a citrate buffer such as sodium citrate. In a specific example, the formulation is an aqueous formulation comprising a saccharide. In a specific example, the saccharide is sucrose. In a specific example, the formulation comprises sucrose, citrate, and a poloxamer, such as poloxamer 188. In one example, the formulation comprises 8-12 mM sodium citrate, 4% to 6% sorbitol (w/v), 0.08% to 0.12% poloxamer 188 (w/v), and a choice Sodium carboxymethylcellulose (such as 0.1 to 1% (w/v)), wherein the formulation has a pH of 5.5 to 7.5.

In another example, the formulation consists of 1 to 50 mg/ml (such as 1 to 20 mg/ml, such as 20 mg/ml, 10 mg/ml, or 5 mg/ml) of the chimeric cells described herein. Hormone protein, such as an IL-1β/IL-1Ra chimeric cytokine protein, such as 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 some instances, the formulation does not contain sodium carboxymethylcellulose. The formulation may comprise 1-20 mg/ml of a chimeric cytokine protein, such as P05. In one example, the formulation consists of 10 mM sodium citrate, 5% sorbitol, 0.1% poloxamer 188, and 1-20 mg/ml P05.

Another specific embodiment is directed to a stable aqueous formulation encapsulated in a BFS container comprising a 1-20 mg/ml chimeric cytokine as described herein, for example, an IL-1β/IL-1Ra inlay A cytokine protein, such as 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 from 5.5 to 7.5. In a specific example, the formulation has a pH of 5.5 to 6.5.

In one example, the BFS container is enclosed in a package, such as an aluminum foil pouch, optionally with an inert gas (e.g., nitrogen) overlay. In one example, the package protects the formulation from photoinduced degradation. In another example, the encapsulating and/or obscuring blanket will protect the formulation from oxidation. The oxidation of the formulation can be assessed using, for example, RP-HPLC.

In a specific example, the formulation is stored in the BFS container at room temperature, such as 25 ° C (eg, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) , 13, 14, 15, 16, 17 or 18 months) is stable. In a specific example, the stability of the formulation is after storage at 2-8 ° C and 60% relative humidity (eg, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 months). The stability of the formulations described herein may be used one or more of the methods to evaluate the metric, such as appearance, pH, absorbance (the A280 or A ₂₈₀₎ 280nm to account for the content of, reduced SDS-PAGE, non-reducing SDS-PAGE, SE-HPLC, RP-HPLC, CIEX-HPLC, and potency. In a specific example, the formulation will meet the specifications disclosed herein after being stored in a BFS container. In a specific example, the formulation has >90% (a/a) monomer when evaluated using SE-HPLC. The abbreviation "(a/a)" means the percentage of the peak area relative to the total area. In a specific example, when evaluated using RP-HPLC, the formulation has 80% (a/a) main peak. In a specific example, when evaluated using CIEX-HPLC, the formulation has 85% (a/a) main peak. In a specific example, the formulation has a form of <10% (a/a) des-Ala when evaluated using CIEX-HPLC. In the specific embodiment, the formulation has an IC ₅₀ of the reference standard IC ₅₀ of 60-140%. In a specific example, the concentration of the formulation is evaluated according to A280 within 10% of its target concentration. In a specific example, a formulation having a nominal concentration of 5 mg/mL is 5 ± 0.5 mg/mL when evaluated according to A280. In a specific example, the pH of the formulation after storage is from 5.7 to 6.3.

In a specific example, the formulation will meet the specifications disclosed herein after being stored in a BFS container, or have better stability than required to meet the specifications. In a specific example, the formulation has >90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (a/a) monomer when evaluated using SE-HPLC. In a specific example, when evaluated using RP-HPLC, the formulation has 80, 81, 82, 83, 84, 85, 86, 87, 87, 89, or 90% (a/a) main peak. In a specific example, when evaluated using CIEX-HPLC, the formulation has 85% (a/a) main peak. In a specific example, the formulation has a form of <10, 9, 8, 7, 6, or 5% (a/a) des-Ala when evaluated using CIEX-HPLC. In a specific example, when evaluated using CIEX-HPLC, the formulation has 5, 4, 3, 2, or 1% methionated species.

In a specific example, when using a light obscuration particle count test to evaluate, 10 μm particles are present in less than or equal to 50 particles per ml, and/or The presence of 25 μm particles per ml of less than or equal to 5 particles indicates that the formulation is stable. In another example, after 4 hours of vortexing of the protein solution at room temperature, for example at 25 °C, the protein monomer form is >90% relative to the aggregated form, indicating that the formulation is stable. The percentage of protein monomer form within the formulation relative to the aggregated form can be assessed using, for example, SEC-HPLC.

In one example, the formulation is stored at the BFS container for at least 12, 13, 14, 15, 16, 17, 18, or 24 months at 2-8 ° C and 60% relative humidity. For stability.

In one example, 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 stored in the BFS container under ambient conditions, for example at room temperature, for example 25 ° C, for at least 6 After the month, the formula was stable.

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 under ambient conditions, for example, at room temperature, such as 25 ° C, after storage in the BFS container for at least 5 months.

In another example, 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 under ambient conditions, for example, at room temperature, such as 25 ° C, after storage in the BFS container for at least 4 months.

According to another example, 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 under ambient conditions, for example, at room temperature, such as 25 ° C, after storage in the BFS container for at least 3 months.

In another example, 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 under ambient conditions, for example at room temperature, such as 25 ° C, after storage in the BFS container for at least 2 months.

In one example, the formulation is stable after storage in the BFS container for at least one month at 2-8 ° C and 60% relative humidity. In another example, the formulation is under ambient conditions, for example The formulation is stable after storage in the BFS container for at least one month at room temperature, for example at 25 °C.

According to one example, for example, when evaluated using a light obscuration particle count test, 10 μm particles have less than or equal to 50 particles per ml, and The presence of 25 μm particles per ml of less than or equal to 5 particles indicates that the formulation is stable. In another example, when evaluated using SE-HPLC, the form of the protein monomer present is >90% relative to the aggregated form, indicating that the formulation is stable. In another example, the formulation is stable based on the primary band conforming to the reference standard in the reduced SDS-PAGE. In another example, the formulation is stable according to the reference band in the non-reduced SDS-PAGE according to the primary band. In another example, when the formulation is evaluated using weak cation exchange HPLC or WCEX-HPLC, a major peak greater than or equal to 85% indicates that the formulation is stable.

According to another example, the formulation comprises P05, and when assessed using weak cation exchange HPLC or WCEX-HPLC, the presence of less than 10% of the des-Ala form of P05 indicates that the formulation is stable. According to another example, the formulation contains P05, and when evaluated using CIEX-HPLC, exists 5, 4, 3, 2, or 1% methionated species, indicating that the formulation is stable.

Also provided herein is a kit comprising a formulation encapsulated in a BFS container as described herein, and optionally including instructions for use.

110‧‧‧reservoir

112‧‧‧ mouth

120‧‧‧Distributor

130‧‧‧Transport system

132‧‧‧ pipeline

132a‧‧‧ pipeline

132b‧‧‧ pipeline

132c‧‧‧ pipeline

134‧‧‧Filter

136‧‧‧buffer tank

138‧‧‧ heat exchanger

140‧‧‧BFS container

210‧‧‧Steps

220‧‧‧Steps

230‧‧‧Steps

240‧‧‧ steps

1 is a block diagram of an embodiment of a BFS package system.

2 is a flow chart of an embodiment of a method of producing a filled BFS container.

Figure 3A is a diagram showing P05 formulated with a phosphate formulation (P05 is 20 mg/ml, 10 mM phosphate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH) 6.5) Dynamic Light Scattering (DLS) results.

Figure 3B is a diagram showing P05 formulated with a citrate formulation (P05 is 20 mg/ml, 10 mM citrate, 5% w/v sorbitol, 0.1% w/v poloxamer 188) , pH 6.0) DLS results.

Figure 4A is a diagram showing the time period during the evaluation using the photoresist particle count test 25μm particles and The number of particles per milliliter of 10 μm particles. The figure also shows the USP<789> specification for particulate matter in the ophthalmic solution, which is 10μm particles per ml 50 particles, and 25μm particles per ml 5 particles.

Figure 4B is a graph showing the number of particles per milliliter of particles of 2 to 10 m during the time period as assessed using a photoresist particle count assay.

Detailed description of the preferred embodiment

Loading a formulation into a blow molded filling seal (BFS container will result in the formulation being exposed to temperatures of approximately 70 ° C to 80 ° C for up to approximately 10 seconds in addition to the mixing associated with the filling process). Surprisingly, the applicant has successfully loaded a formulation into a BFS container, as soon as it is packaged into a BFS container, and at BFS. The stability of the formulation is exhibited in the case of both extended storage periods within the container.

Aspects and specific examples are directed to BFS processing to produce a stable formulation in a BFS container. Additional aspects and specific examples are directed to a formulation that is stable in a BFS container. Typically, the formulation is for ocular delivery, such as for topical ocular delivery.

As used herein, the term "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 WO 2014/160371 and US 2014/0308239. A formulation as described herein is aqueous and includes 10 mM sodium citrate, pH 6.0, 5% sorbitol (w/v) and 0.1% poloxamer (w/v).

Referring to Figure 1, a block diagram of an example of a BFS package system in accordance with one embodiment is illustrated. The system includes a reservoir 110 containing the formulation, a BFS filling system or dispenser 120, and a delivery system 130 that transfers the formulation from the reservoir 110 to the dispenser 120. The reservoir 110 can be used to store the formulation for a short period of time or an extended period of time to accommodate the manufacturing schedule. Those skilled in the art will appreciate that the formulation can be prepared for use in a BFS process prior to being placed in the reservoir 110, and that the preparation can include setting the concentration and end of the drug substance to be supplied to the formulation. Where the desired concentration of the product is different, a formulation buffer is added and the formulation is selectively filtered to reduce the bioburden level. The dispenser 120 is configured to fill a plurality of BFS containers with the formulation according to conventional BFS filling techniques. 140. After filling, the BFS containers 140 are sealed in accordance with conventional BFS processes and techniques.

As discussed above, the high temperatures that the formulation may be exposed during the BFS process raise concerns about the formulation stability of the package. Thus, certain aspects are directed to cooling the formulation and maintaining it in practical ice cold before dispensing the formulation into the BFS containers 140. In one embodiment, the formulation can be maintained in the storage tank 110 at a temperature in the range of 2-8 °C. Therefore, the storage tank 110 can be insulated. In some examples, the storage tank 110 can be actively cooled using, for example, a circulating refrigerant supplied via a port 112. Methods of insulation and cooling are known in the art. In such instances, the formulation that has been cooled can be provided to the storage tank 110, or the formulation can be cooled in the storage tank by a slightly elevated temperature.

To further transfer the formulation from the storage tank 110 to the dispenser 120, the formulation is maintained within the desired cooling temperature range, so that at least a portion of the delivery system 130 can be insulated and/or actively cooled. In one embodiment, the delivery system 130 includes lines 132 (for example, such as tubes, pipes, or hoses) that flow from the reservoir 110 to the reservoir via a line. The dispenser 120. As shown in FIG. 1, in some embodiments, the delivery system optionally further includes a filter 134, a buffer tank 136, and/or a heat exchanger 138. The filter 134 can be, for example, a sterile filter. In such instances, at least some of the segments 132, such as to direct the formulation to various components of the delivery system 130 and various components exiting the delivery system 130, may be Insulated or cooled. For example, the delivery system 130 can include a first length of cooled or insulated line 132a that transports the formulation from the cooled and/or insulated storage tank 110 to the filter 134, and a second stage of cooling/insulation Line 132b, which transports the formulation from the heat exchanger 138 to the distributor 120. A third length of line 132c directs the formulation from the buffer tank 136 to the heat exchanger 138, which may also be insulated and/or cooled. Such cooled and/or insulated lines may include blue NPD hoses, white-wrapped hoses, or insulated jacketed or foamed tubing.

The buffer tank 136 can be used, for example, to control the volume and/or flow rate of the formulation supplied to the dispenser 120. In case any heating occurs during the transfer of the formulation from the storage tank 110 to the heat exchanger, the heat exchanger 138 can be used to cool the formulation before the formulation reaches the dispenser. For example, the formulation may become slightly warmer during storage in the buffer tank. Thus, the heat exchanger 138 can be used to lower the temperature of the formulation. In another example, the heat exchanger 138 can circulate refrigerant around the lines 132a, 132b, and 132c within the delivery system 130 to cool the lines, and thereby cool the formulation flowing through the lines.

In one embodiment, a thermocouple 150 can be coupled to the line segments 132b just prior to the formulation entering the dispenser so that the formulation can be measured just prior to dispensing the formulation into the BFS containers 140. temperature. This information can be used to adjust the cooling profile of the system to control the temperature of the formulation. For example, if the measured temperature of the formulation is above a desired temperature range, the storage tank 110 and/or heat exchanger 138 can be adjusted to further cool the formulation.

Thus, in accordance with certain embodiments, the temperature of the formulation can be controlled prior to the filling step of the BFS process by providing a stable (e.g., insulating) mechanism of cooling and/or temperature within the delivery system 130. By ensuring that the formulation is at a relatively low temperature (eg, at 15 ° C or lower, at 10 ° C or lower, or at 8 ° C or lower) when dispensed into the BFS containers. The effect of contacting a warm BFS container can be minimized, resulting in a stable package formulation. Exemplary formulations and formulation components are described herein.

In some embodiments, the formulation comprises a chimeric cytokine protein as described herein at a concentration ranging from 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. Unless otherwise expressly stated, when used in reference herein, the term includes the range.

Without wishing to be bound by theory, in some cases, the combination of components and/or components included in the formulation itself may protect against exposure to elevated temperatures for destabilization. The formulation may include a buffer which also provides increased stability and resistance to temperature effects, as demonstrated in the examples provided below. In particular, the citrate (sodium citrate) buffer increases the temperature of the unfolding of the chimeric cytokine protein within the exemplary formulation by more than the phosphate buffer (sodium phosphate). 10 ° C (see Example 1). In a specific example, the formulation includes sodium citrate. In a specific example, the chimeric cytokine protein in the formulation begins to unfold at a temperature of at least 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 °C.

In one embodiment, a chimeric cytokine protein, such as P05, as described herein is formulated to a concentration of 5 mg/ml to 20 mg/ml (eg, 1 mg/ml, 5 mg/ml, or 20 mg/ml). Concentration), sodium citrate formulated at pH 5.5-6.5 (such as pH 6.0), 5-15 mM (such as 10 mM), containing 2.5-7.5% (such as 5%) w/v sorbitol and 0.05- 0.15% (such as 0.1%) w/v poloxamer, such as poloxamer 188 (also known as, for example, Lutrol® F-68 or Kolliphor® P 188).

In a specific example, the chimeric cytokine protein is selected from one or more of the group consisting of P01, P02, P03, P04, P05, P06, and P07. In a specific example, the chimeric cytokine is P05.

In particular embodiments, the components of the formulations as described herein may be present in amounts ranging from about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or It is 50%. In a particular embodiment, the components of one formulation are present in amounts varying by about 10% as provided herein.

In a specific example, 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.

In a specific example, 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.

In a specific example, the formulation contains 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 of poloxamer 188.

In a specific example, the concentration of the chimeric cytokine protein (such as P05) in the formulation is 1-50 mg/ml, 1-25 mg/ml, or 1-20 mg/ml. In a specific example, 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 a specific example, the pH of the formulation is from 5.5 to 7.5, or from 5.5 to 6.5.

In a specific example, 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 inlay. A cytokine protein, such as P05. In a specific example, the pH of the formulation is from 5.5 to 7.5. In a specific example, the pH is from 5.5 to 6.5. In a specific example, the pH is from 6 to 7.

In a specific example, 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. A chimeric cytokine protein, such as P05. In a specific example, the pH of the formulation is from 5.5 to 7.5. In a specific example, the pH is from 5.5 to 6.5. In a specific example, the pH is from 6 to 7.

In one embodiment, the formulation comprises 10 mM sodium citrate, pH 6.0, containing 5% w/v sorbitol and 0.1% w/v poloxamer, such as poloxamer 188. In a specific example, the amount of the formulation component can vary by about 5% in the values provided herein. 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In a specific example, the pH of the formulation is from 5.5 to 7.5. In a specific example, the pH is from 5.5 to 6.5. In a specific example, the pH is from 6 to 7.

Topical eye medications are generally self-administered by the patient. Because the patient may store the drug for a relatively long period of time, the formulation may experience higher temperatures and greater levels of agitation stress than formulations typically only stored 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. Stirring stress can cause precipitation and thermal stress can lead to precipitation and chemical degradation. In addition, thermal stress may be exposed during loading of the compound to a BFS delivery device. The formulations used herein provide outstanding stability when exposed to agitation stress and heat.

Typically, the formulations mentioned herein are stable. In a specific example, the formulation exhibits stability immediately after dispensing into the BFS container and/or after storage in the BFS container. Exemplary conditions and measurement systems that can be used to assess stability are discussed in more detail below.

In a specific embodiment, the formulation in the BFS container is stable from 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; Day; 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 Month, four Months, five months, six months, seven months, eight months, or longer.

It can be stored, for example at 2-8 ° C, for example at least 2, 4, 6, 8, 12, 15, 16, 17, or 18 months, or under ambient conditions, such as at room temperature (RT) For example, at about 25 ° C, storage duration such as at least two weeks, one month, two months, three months, five months, six months, twelve months, 13 months, 14 months, 15 Stability was assessed monthly, 16 months, 17 months, or 18 months later. In a specific example, the formulation is stable at 2-8 ° C for at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months. In a specific example, the formulation is stable after exposure to room temperature for at least 3, 4, 5, or 6 months. In some such specific examples, the formulation is stored in a BFS container, such as at least 3, 4, 5, 6, 7.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 It is stable after a month.

Stability can be assessed, for example, based on methods and criteria described herein or known in the art. For example, the assessment of stability can be based on physical purity (eg, no aggregation, such as when using particle size screening HPLC, also referred to herein as particle size analysis, SE HPLC, or SEC HPLC), chemical purity ( For example, when using weak cation exchange HPLC, reverse phase HPLC, and/or SDS PAGE (eg, reduced or non-reduced SDS PAGE), and/or particle size (eg, when visually assessed or by use) HIAC liquid particle counter (Beckman Coulter, Brea, CA) for particle counting).

Stability can also be assessed based on visual appearance. In a specific example, if the formulation is substantially non-visible, the clarification of the particles is slightly whiter. The solution is stable with a colored solution.

In a specific example, the proof of stability is based on guidelines for adhering to particulate matter in ophthalmic solutions, such as those set forth in USP < 789> (U.S. Pharmacopeia, Particulate Matter in Opthalmic Solutions).

In a specific example, the formulation is evaluated, for example, using a photoresist particle count assay (such as a photoresist particle count assay as described in USP <788>). 10 μm particles have less than or equal to 50 particles per milliliter, and/or The 25 μm particles have less than or equal to 50 particles per milliliter.

In a specific example, the formulation is evaluated, for example, using a microscopic particle count assay (such as a microscopic particle count assay as described in USP <788>). 10 μm particles have less than or equal to 50 particles per milliliter, and/or The 25 μm particles have less than or equal to 5 particles per milliliter.

In a specific example, the proof of stability is based on guidelines for adhering to particulate matter in the injectable solution, such as those set forth in USP <788> (U.S. Pharmacopeia, Particulate Matter in Injections).

In a specific example, the formulation is evaluated, for example, using a photoresist particle count assay (such as a photoresist particle count assay as described in USP <788>). 10 μm particles have less than or equal to 6000 particles per container (the container has a volume of 100 ml or less), and/or The 25 μm particles have less than or equal to 600 particles per container (the container has a volume of 100 ml or less).

In a specific example, the formulation is evaluated, for example, using a microscopic particle count assay (such as a microscopic particle count assay as described in USP <788>). 10 μm particles have less than or equal to 3,000 particles per 5 ml, and/or The 25 μm particles have less than or equal to 300 particles per 5 ml.

In a specific example, the protein in one formulation is resistant to agitation stress, such as when the protein solution vortexes at room temperature (RT) for 1-8 hours, for example, after vortexing for 4 hours at RT, there is no Aggregation (no aggregation can be expressed, for example, if the formula contains >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%) , or >99% of the protein monomer form, relative to the aggregated form). Aggregation can be assessed, for example, using the methods described herein or methods known in the art. For example, aggregation can be assessed using ultracentrifugation, particle size exclusion chromatography, gel electrophoresis, dynamic light scattering, and/or turbidity measurement.

In some aspects, stability is analyzed by physical or chemical methods known in the art. For example, physical purity or no aggregation can be determined using particle size screening HPLC or other methods that determine the relative amount of monomeric polypeptide within the formulation. Typically, a formulation with acceptable stability contains >90% of the monomeric form of the therapeutic protein (such as a chimeric cytokine, such as P05), relative to the form in which the protein is aggregated. In a specific example, the formulation contains >90% (such as >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99) %) a monomeric form of the therapeutic protein (such as a chimeric cytokine, such as P05) relative to the form in which the protein is aggregated.

Chemical purity can be used with weak cation exchange HPLC or reverse phase Determined by HPLC. Typically, a formulation with acceptable stability, such as when used to evaluate weak cation exchange HPLC, contains >80% of the natural molecule relative to the chemically modified form of the molecule. In a specific example, the formulation contains >80% (such as >85%, >87%, >90%, or >95%) of the natural molecule relative to the chemically modified form of the molecule (eg, oxidation or B) Degenerate form).

Particles can be visually identified. In a specific example, the formulation is a formulation of substantially non-visually identifiable particles.

Biologic treatments may be problematic for administration because they have a relatively short shelf life or require special storage conditions that can impair storage, shipping, and patient use, as well as ensuring adequate supply of biological agents. An advantage of certain formulations provided herein is that the formulation is surprisingly stable, not only under freezing conditions, but also at room temperature (such as 25 ° C) and temperatures above room temperature (such as 40 ° C). Thus, the formulation is typically in the form of a liquid that is stable at room temperature (RT) (such as at 25 ° C) for a period of at least three days, five days, one week, ten days, two weeks, three Week, 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 longer. In a specific example, one month is determined on a date-to-date basis, such as from the first day of the month to the first day of the second month.

In other aspects, the formulation is stable from 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 at least two days. Period; 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 longer.

In one example, when a protein component of a formulation, such as P05, has a concentration of 20 mg/ml, the formulation is stable at 25 ° C for one month and at 40 ° C for one week.

In another specific embodiment, a formulation comprising a protein, such as P05, at a concentration of 1-20 mg/ml, such as about 1 mg/ml, 5 mg/ml, or 10 mg/ml, the formulation is at least 25 ° C for at least Three months is stable. In some specific examples, the formulation is stable for at least five months.

In a specific example, one comprises 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. Formulating at a temperature of from 2 ° C to 8 ° C and/or at room temperature, for example at 25 ° C for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 Or 18 months, it is stable. In some specific examples, a formulation consisting of 10 mM Na citric acid, pH 6.0, 5% sorbitol, 0.1% poloxamer 188, and 5 mg/ml or 20 mg/ml P05 at 2 ° C At least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, and/or at room temperature, at 8 ° C For example, at 25 ° C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months is stable. In some specific examples, a formulation consisting of 10 mM Na citric acid, pH 6.0, 5% sorbitol, 0.1% poloxamer 188, and 5 mg/ml or 20 mg/ml P05 at 2 ° C It is stable for at least five months up to 8 ° C and/or at room temperature, for example at 25 ° C for at least five months.

The aspects and specific examples are directed to a formulation that is stable in a BFS container, and a method of preparing a stable formulation in a BFS container using a BFS process. Figure 2 is a flow chart of an example of such a method. The first step 210 includes using a BFS process to prepare and cool the formulation to be packaged. As discussed above, this step 210 can include providing a bulk drug substance (such as P05), adding a desired formulation component, such as a formulation buffer (such as a citrate buffer as discussed above) to the production facility. The formulation is cooled to the desired temperature range in the storage tank 110 or before the formulation is placed in the storage tank. Step 220 includes transmitting the recipe to the BFS distributor via the BFS delivery system 130.

As discussed above, the conveyor system 130 can include components, such as cooled and/or insulated pipelines 132a, 132b, and 132c, and/or active cooling mechanisms, such as heat exchanger 138. These cooling components can be configured to cool and/or maintain the formulation within the desired temperature range. The BFS container can then be filled with the formulation (step 230) and sealed using a conventional BFS process (step 240). As discussed above, surprisingly, using the formulation as discussed herein, combining the delivery system 130 including the cooling components, a BFS process can be used to provide a stable packaged formulation, with knowledge based on the artisan tradition. The opposite is expected.

Having described several aspects of at least one specific example, it will be appreciated that various changes, modifications, and improvements are readily apparent to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure, and are intended to fall within the scope of the invention. It will also be appreciated that the specific examples of the methods and apparatus discussed herein are not limited to the application of the details and the configuration of the components set forth in the following description or illustrated in the drawings. The methods and apparatus can be implemented in other specific embodiments and performed or performed in various ways. The examples of specific implementations provided herein are for illustrative purposes only and are not intended to be limiting. Also, the phrases and terms used herein are for the purpose of description and should not be considered as limiting. The use of "including", "comprising", "having", "containing", "involving", and the like are intended to encompass the items listed and Equal to it. References to "or" may be construed as inclusive, and any term that is recited with the use of "or" can mean any of the singular, more than one, and all of the terms.

Chimeric cytokine protein

The formulations referred to herein are typically aqueous formulations. The formulation referred to herein includes a chimeric cytokine protein.

As used herein, "a chimeric cytokine protein" refers to a protein comprising an IL-1 family chimeric cytokine region as described in WO 2012/016203 or WO 2012/103240, etc. The entire content is hereby incorporated by reference. The chimeric cytokine protein comprises a cytokine domain of a chimeric interleukin-1 (IL-1) family, wherein at least one first segment of the region is At least 20 amino acids, and having at least 80% amino acid identity to a corresponding segment of the first IL-1 family cytokine, and at least one second segment of the region having a length of at least 20 amino acids and at least 80% amino acid identity to the corresponding segment of the second IL-1 family cytokine. In a specific example, 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. In a specific example, the first and second IL-1 family cytokines are selected from the group consisting of IL-1β, IL-1α, and IL-1Ra. In a specific example, the first and second IL-1 family cytokines are IL-1β and IL-1Ra, respectively. In a specific example, the protein consists of a single polypeptide chain of 150-160 amino acids in length.

Typically, the chimeric cytokine protein acts as an IL-1 inhibitor. In a specific example, the chimeric cytokine region binds to IL-1 receptor I (IL-1RI) (eg, has a Kd of less than 5 nM). In a specific example, the chimeric cytokine region inhibits the activity of IL-1RI. In a specific example, the chimeric cytokine region is inhibited by IL-1β at a concentration of 0.1 ng/ml, and the IC50 is less than 50 nM. In a specific example, the protein inhibits IL-1β-induced IL-6 expression in MG-63 cells.

In a specific example, the chimeric cytokine region is not naturally occurring. In a specific example, the chimeric cytokine region is associated with human IL-1Ra (SEQ ID NO: 18), human IL-1β (SEQ ID NO: 16 and/or human IL-1α (SEQ ID NO: 17) The identity is less than 80%.

The amino acid sequence of IL-1β (human) referred to herein is: (Sequence identification number: 16).

The amino acid sequence of IL-1α (human) referred to herein is: (Sequence identification number: 17).

The amino acid sequence of IL-1Ra (human) referred to herein is: (Sequence identification number: 18).

As used herein, the terms IL-1β, IL-1α, and IL-1Ra refer to the respective mature proteins.

In a specific example, the cytokine region comprises at least two non-contiguous segments of at least 20 amino acids in length that are at least 90% identical to respective corresponding segments of human IL-1 β. In a specific example, the two non-contiguous segments are identical to the respective corresponding segments of human IL-1β. In a specific example, the two non-contiguous segments are between 25-40 amino acids in length. In a specific example, an amine that is not in the two discontinuous segments The amino acids not in the two discontinuous segments are at least 90% identical to human IL-1Ra.

In a specific example, the chimeric cytokine protein is an IL-1β/IL-1Ra chimeric cytokine protein, such as P05.

In a specific example, the formulation referred to herein comprises a protein or polypeptide comprising the following or consisting of: P01 (SEQ ID NO: 1), P02 (SEQ ID NO: 2), P03 (SEQ ID NO: :3), P04 (sequence identification number: 4), P05 (sequence identification number: 5), P06 (sequence identification number: 6), P07 (sequence identification number: 7), P08 (sequence identification number: 8), P09 (sequence identification number: 9), P10 (sequence identification number: 10), P11 sequence identification number: 11), P12 sequence identification number: 12), P13 sequence identification number: 13), P14 (sequence identification number: 14), Or the serial identification number: 15. These sequences are provided below.

In a specific example, the formulation referred to herein comprises an IL-1β/IL-1Ra chimeric cytokine protein comprising the following or consisting of: P01 (SEQ ID NO: 1), P02 (SEQ IDENTIFICATION No.: 2), P03 (sequence identification number: 3), P04 (sequence identification number: 4), P05 (sequence identification number: 5), P06 (sequence identification number: 6), or P07 (sequence identification number: 7) ).

In a specific example, the formulation referred to herein comprises a protein comprising a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to: P01 ( Sequence identification number: 1), P02 (sequence identification number: 2), P03 (sequence identification number: 3), P04 (sequence identification number: 4), P05 (sequence identification number: 5), P06 (sequence identification number: 6) ), P07 (sequence identification number: 7), P08 (sequence identification number: 8), P09 (sequence identification number: 9), P10 (sequence identification number: 10), P11 (sequence identification number: 11), P12 sequence identification number: 12), P13 sequence identification number: 13), P14 (sequence identification number: 14), or sequence identification number: 15.

In a specific example, the chimeric cytokine protein differs from the following by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid: P01 (SEQ ID NO: 1), P02 (sequence identification number: 2), P03 (sequence identification number: 3), P04 (sequence identification number: 4), P05 (sequence identification number: 5), P06 (sequence identification number: 6), P07 ( Sequence identification number: 7), P08 (sequence identification number: 8), P09 (sequence identification number: 9), P10 (sequence identification number: 10), P11 (sequence identification number: 11), P12 sequence identification number: 12) , P13 sequence identification number: 13), P14 (sequence identification number: 14), or sequence identification number: 15.

In a specific example, the formulation referred to herein includes a protein comprising P01 (SEQ ID NO: 1), P02 (SEQ ID NO: 2), P03 (SEQ ID NO: 3), P04 (SEQ ID NO: 3) :4), or P05 (sequence identification number: 5) has a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity.

In a specific example, the formulation referred to herein includes a protein comprising P01 (SEQ ID NO: 1), P02 (SEQ ID NO: 2), P03 (SEQ ID NO: 3), P04 (SEQ ID NO: 3) :4), or P05 (sequence identification number: 5) has a sequence of at least 95% identity. In a specific example, the formulation referred to herein comprises a protein comprising a sequence that is at least 95% identical to P01 (SEQ ID NO: 1). In the specific case, this article The formulation referred to includes a protein comprising a sequence that is at least 95% identical to P02 (SEQ ID NO: 2). In a specific example, the formulation referred to herein comprises a protein comprising a sequence that is at least 95% identical to P03 (SEQ ID NO: 3). In a specific example, the formulation referred to herein comprises a protein comprising a sequence that is at least 95% identical to P04 (SEQ ID NO: 4). In a specific example, the formulation referred to herein comprises a protein comprising a sequence that is at least 95% identical to P05 (SEQ ID NO: 5).

In a specific example, the chimeric cytokine protein is more thermostable than human IL-1Ra and human IL-1β. In a specific example, the chimeric cytokine protein comprises an IL-1β/IL-1Ra chimeric cytokine region having a higher thermostability than human IL-1Ra and human IL-1β. In a specific example, the formulation comprises a chimeric cytokine protein having a chimeric IL-1β/IL-1Ra cytokine region comprising, and thermally stable, a chimeric IL-1β/IL-1Ra cytokine region One or more structural components associated with sex, such as a unique salt bridge or hydrogen bond, as described in Example 10 of WO 2012/016203. In some embodiments, the chimeric cytokine region comprises one or more of the following (such as 3, 1, or all 3): (i) E44 from human IL-1Ra and from human IL K65 of -1β; (ii) R14 from human IL-1Ra and Q149 from human IL-1β; and (iii) S152 from human IL-1β and K45 from human IL-1Ra. In some embodiments, the IL-1β/IL-1Ra chimeric cytokine protein comprises or consists of: a sequence, and P03 (SEQ ID NO: 3), P04 (SEQ ID NO: 4) , or P05 (sequence identification number: 5) A sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity. In some embodiments, the chimeric cytokine protein region comprises or consists of: P03 (SEQ ID NO: 3), P04 (SEQ ID NO: 4), or P05 (SEQ ID NO: :5) is the same sequence.

In the particular embodiment, the chimeric cytokines region exhibits the following thermal properties of one, two, three, four of, or all of: (i) having a melting temperature (T _m) than human IL-1β and human IL-1Ra is at least 2 ℃, (ii) at least 58 deg.] C having a T _m (iii) has a higher than human IL-1Ra and IL-1β in human T _m, (iv) than human IL-1Ra And a higher unfolding temperature of human IL-l[beta], and (v) an unfolding temperature of at least 50[deg.] C., for example, using differential scanning fluorimetry. The concentration in the PBS formulated with pH 7.4 was evaluated to be 0.5 mg/mL, for example as described in Example 7 of WO 2012/016203.

In a specific example, the chimeric cytokine protein comprises P05 (SEQ ID NO: 5) or P05 (SEQ ID NO: 5).

Use, treatment, and kit

In a particular embodiment, one of the formulations described herein is for topical administration, such as topical eye administration. In a specific example, the formulation is for administration such as eye drops. In a specific example, the formulation is administered by a subject. In a specific example, a method of treatment described herein comprises self-administering the formulation by a subject. In a specific example, the BFS container is suitable for administration as a drug (such as self-injection The drug delivery device of the formulation, for example, is administered topically to the eye, such as, for example, an eye drop.

In a specific example, the protein is effective in modulating an immune or inflammatory response. In a specific example, the formulation is for use in modulating an immune or inflammatory response. Also provided herein is a method of modulating an immune or inflammatory response of a subject, such as a human or non-human animal, comprising administering a formulation described herein to the subject.

In a specific example, the protein is effective in treating diseases of the eye. In a specific example, the formulation is for the treatment of ocular diseases. Also provided herein is a method of treating a subject's ocular condition comprising administering a formulation described herein to the subject. In a specific example, the eye disease is dry eye disease or allergic conjunctivitis.

In a specific example, the protein is effective in the treatment of allergic rhinitis. In a specific example, the formulation is for the treatment of allergic rhinitis. Also provided herein is a method of treating a subject's allergic rhinitis comprising administering a formulation described herein to the subject.

In one embodiment, provided herein is a method of treating a subject, such as an ocular condition (such as dry eye or dry eye disorder), the method comprising administering (eg, topically administering) a formulation described herein to the subject . In a specific example, the primary system self-administers the formulation, for example by topical application of the formulation, such as to the eye, such as, for example, an eye drop.

Also provided herein is a kit comprising a formulation in a BFS container as described herein, and optionally including instructions for use. In a specific example, the container is suitable for administration as a drug (such as self-administration) The delivery device is, for example, for topical administration, such as for topical application to the eye, such as, for example, eye drops.

The sequences of exemplary chimeric proteins that can be included in the formulations described herein are provided below.

Additional exemplary chimeric IL-1 family proteins also include the following:

The polypeptide below is a chimeric cell-exciting region comprising at least two segments from IL-1α and at least two segments from IL-1Ra.

All of the patents, published patent applications, and published references cited herein are hereby incorporated by reference for all purposes.

The functions and advantages of the foregoing and other specific examples will be more fully understood from the following description. The embodiments are merely intended to be illustrative in nature and are not considered as limiting the scope of the systems and methods discussed herein.

Example Example 1: Stability of P05 in phosphate and citrate buffers

Dynamic light scattering or DLS (also known as quasi-elastic light scattering or QELS) measures the diffusion of an analyte (such as P05) in a well plate by focusing the laser light onto the sample and A fast photon counter measures to monitor the rate of fluctuation of scattered light. Using a mathematical technique, called a correlation function, to quantify the rate of fluctuation to determine the diffusion system number. The hydration radius (Rh) is obtained using a diffusion coefficient according to the Stokes-Einstein equation.

P05 radius is measured as a flat disk reader DLS ^{(Wyatt DynaPro TM, Wyatt Technologies,} Santa Barbara, CA) in the temperature rise function. The capture time is 5 seconds and 5 scans are performed per measurement. The load rate was 0.17 ° C / min. As the protein unfolds, the radius increases. The temperature at which the radius increases is referred to as _Ton (the temperature at which unfolding).

The experiment was performed on 20 mg/mL of P05 in two formulations: (i) 10 mM phosphate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.5 And (ii) 10 mM sodium citrate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.0. The results depicted in Figures 3A and 3B show that the temperature at which _Ton occurs in the citrate buffer is higher relative to the phosphate buffer. The _Ton of P05 in the citrate buffer was 48.2 °C, and the _Ton of P05 in the phosphate buffer was 35.2 °C. This large difference in _Ton is surprising and indicates that P05 in the citrate buffer is more stable than phosphate buffer.

Thus, in some specific examples herein, one formulation comprises a citrate buffer.

Example 2: Stability of a formulation in a blow molded filled sealed container

Experiments were conducted to investigate the effect of encapsulating P05 and subsequent storage in an aluminum foil pouch with or without a nitrogen blanket, in a blow molded fill seal (BFS) container. For formulations containing active drug, P05 to perform the test. The bulk drug substance is formulated into an aqueous solution containing 10 mM sodium citrate. 5% w/v sorbitol, 0.1% w/v poloxamer 188, for blow molding filling at pH 6.0. The concentration of P05 is 5.0 mg/mL.

The number of filled units is approximately 1000 containers and the nominal fill volume is 0.32 mL. The process stream is insulated and cooled prior to entering the blow molding apparatus by maintaining the process stream at 2-8 ° C using a method as described herein, the method of insulating and cooling including the storage The pipeline between the tank and the distributor is insulated, and a heat exchanger is used between the buffer tank and the distributor. A portion of the container is loaded into foil packages with a nitrogen blanket. The initial characterization was performed after encapsulation into the BFS container and further evaluated for stability after storage at two temperatures (2 to 8 ° C and 25 ° C) with or without bagging.

Analysis of the initial characteristics include: depending on the concentration of A _280, SDS-PAGE, SEC-HPLC , wCEX-HPLC, RP-HPLC, osmolality, and particle analysis photoresist. The stability of P05 was monitored monthly by SEC-HPLC, wCEX-HPLC and RP-HPLC. The evaluation of A _{280 and} the evaluation of pH and osmotic pressure were performed at 5, 12, and 18 months.

Characterization of the initial P05 formulation after processing of the blow molding filling shows retention of stability

The initial analysis of P05 after the processing of the blow molding filling proved that the protein retains its chemical and physical stability despite the processing of the blow molding filling. P05 formulation not exposed to the BFS process (aqueous formulation containing P05 at a concentration of 50 mg/mL, 0.01% w/v poloxamer 188, 5% w/v sorbitol, 10 mM sodium citrate , at pH 6.5), and the P05 formulation of the process undergoing the blow molding filling (Aqueous formulation containing P05 at a concentration of 5 mg/mL, 0.1% w/v poloxamer 188; 5% w/v sorbitol; 10 mM sodium citrate at pH 6.0) for comparison. These formulations were made using the same production batch of P05.

The results show that at the time zero after the blow molding filling package, the blow molded filling formulation retains outstanding stability even after exposure to potentially harmful blow molding filling processes, as is done by using particle size screening. Chromatography, RP-HPLC, wCEX, and SDS-PAGE (see Table 2) are indicated.

In addition to the above analysis, a light obscuration can be used according to the method USP<789> to perform particle analysis on a recipe packaged in a BFS container. The results and specifications of this method are shown in Table 3 below. The subvisible particle counts under the microscope fall within the specifications of the topical eye drug of the USP and are consistent with the lack of visible precipitation in the BFS container.

In case of a slight difference effect, depending on when the container for the blow molding filling operation is filled, the formulation of the early, intermediate and late (time) partially filled containers during the operation is compared (for example, to study whether the equipment is in time) Heating during the period is an adverse effect on the formulation). The results are shown in Table 3. The number of subvisible particles observed under the microscope did not increase over time and in fact also tends to decrease over time.

These data demonstrate that P05 is physically stable (according to SEC-HPLC, light-shielding, and visual observation) and chemically stable (based on wCEX-HPLC, RP-HPLC) immediately after processing of the blow molding fill. , SDS-PAGE measurement).

The osmotic pressure is measured, as well as the concentration of A ₂₈₀ as part of the initial characterization. The average is a measure of the osmotic pressure of 317mOsm, and an average concentration based on A ₂₈₀ is a measure of 5.0mg / mL.

P05 formulation retention stability in a blow molded container

The P05 formulation in the BFS container was stored in an incubator at 25 ° C and 60% relative humidity or 2 to 8 ° C. Samples were analyzed by SEC, RP-HPLC, and wCEX-HPLC every other month. SEC separates the monomer from the aggregate; the formation of aggregates results in a decrease in the main peak. Separation is provided based on hydrophobicity; oxidation of the protein, such as oxidation of methionine, results in additional peaks. wCEX-HPLC measures the formation of charge variants (deamination leads to additional peaks). At the 4th and 5th months, the concentration of A ₂₈₀ was measured. At 5 months, 12 months, and 18 months, osmotic pressure (to assess evaporation), pH, and concentration according to A ₂₈₀ were also measured.

The results of SEC-HPLC (see Table 4) indicate that P05 was stored in a BFS container at room temperature (25 ° C) for at least 6 months, or 2 to 8 ° C for at least 18 months, without forming aggregates. The bagged vial does not affect the physical stability of the product at any temperature after nitrogen flushing.

The stability results of the wCEX-HPLC of the P05 formulation are shown in Tables 5 and 6 (% of the main peak and % of the deamidamine, respectively).

The results of wCEX-HPLC indicate that the P05 formulation in the blow molded filled sealed container is stable for up to five months at room temperature. P05 at 2 Stability was also retained for at least 18 months at 8 °C. The bagged container does not affect the stability of the product at any temperature after nitrogen flushing.

The stability results of the RP-HPLC of the P05 formulation in the blow molded filled sealed container are shown in Tables 7 and 8 (% of the main peak and % of the protein peak of oxidation, respectively).

The results of RP-HPLC indicate that the P05 formulation stored in the BFS container was stable at room temperature (25 ° C) for at least 6 months and at 2 to 8 ° C for at least 18 months.

In addition, osmotic pressure and pH measurements indicated that the osmotic pressure or pH of the sample at 2 to 8 ° C or 25 ° C did not change significantly during the time (see Table 9). This indicates that little or no evaporation occurs and the pH of the solution is still stable. The protein concentration assessed using A _{280 was} also consistent with previous measurements (see Table 9). In general, EBI-005 exhibits outstanding physical stability at 2 to 8 ° C and at ambient temperature (RT) after prolonged storage in a blow molded filled vial.

* The vehicle is an aqueous solution comprising the same formulation components, except that it does not contain P05.

Example 3: Long-term stability of clinical drugs in a blow molded filling sealed container

Production of P05 (adapted to a concentration of 5 mg / ml, formulated with 10 mM sodium citrate, 5% w / v sorbitol, 0.1% poloxamer 188, pH 6.0 aqueous solution) for clinical trials, packaged in The air-molded filling and sealing container as described in Embodiment 2 is maintained under the control conditions according to the current good manufacturing practices (cGMP). In order to evaluate the temperature of the formulation immediately prior to dispensing into the BFS container, a thermocouple was placed close to the filling system (dispenser) so that the temperature of the formulation during filling could be evaluated. The thermocouple indicated a temperature of 8 ° C during the filling and a thermocouple of the bulk solution in the tank (reservoir) indicated a temperature of 3 ° C.

Table 10-13 provides the results of the stability of the cGMP batch of formulated drugs, referred to herein as "X batch" and "Y batch". The data for the 25 °C stability of the second batch is shown first (see Tables 10 and 11), followed by the data stored at 2 to 8 °C (see Tables 12 and 13). Evaluations included appearance, pH, content in A280, reduced SDS-PAGE, non-reduced SDS-PAGE, SE-HPLC, RP-HPLC, CIEX-HPLC, and potency. SE-HPL (also referred to herein as SEC) separates monomers and aggregates; formation of aggregates results in a decrease in the main peak. RP-HPLC provides separation based on hydrophobicity; oxidation of the protein (such as oxidation of methionine) results in a decrease in the main peak. CIEX-HPLC (cation exchange HPLC) separates the charge variants of proteins (which are caused, for example, by aspartic acid) Deamination acts to form aspartic acid, or deamidamine of glutamic acid to form glutamic acid; the formation of a charge variant results in a decrease in the main peak.

NS=unsampling

NS=unsampling

NS=unsampling

NS=unsampling

The results shown in Tables 10-13 confirm the results of Example 2. These results demonstrate that the P05 formulation is still stable and meets specifications for at least 6 months at 25 °C and at least 12 months at 2 to 8 °C.

The additional data for the X batch was collected by light obscuration analysis according to the method USP <789> for measuring the particles in the local eye drug formulation. This method measures the number of total particles in three sizes ( 10 microns, 25 microns, and 2 to 10 microns). on 10 micron and The specifications of 25 microns (specified by USP) are shown in Table 14 and Figure 4A. Particles in the 2 to 10 micron range are not specified. The results shown in Figures 4A and 4B demonstrate that the initial increase in the number of subvisible particles under the microscope (between time = 0 and 11 months), the number of particles remains consistent, and is stored in 2 to 8 ° C for up to 17 months did not exceed the specifications.

Applicants have discovered that the improved chimeric cytokine formulation in a blow molded, sealed sealed container can be produced using the improved methods and formulations provided herein. Again, these results demonstrate that the formulation is stable after prolonged storage, for example, at room temperature for at least 6 months, and at 2 to 8 °C for at least 17 months.

The foregoing description and drawings are intended to be illustrative only, and the scope of the invention

<110> Elaine Biotherapy Co., Ltd.

<120> A chimeric cytokine protein formulation that is stable in a blow molded, sealed, sealed container. method

<130> D2046-7058WO

<140>

<141>

<150> 61/952,701

<151> 2014-03-13

<160> 18

<170> PatentIn version 3.5

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Claims (30)

A method of providing a stable formulation in a blow molded fill seal (BFS) container, the formulation comprising a chimeric cytokine protein, the method comprising dispensing the formulation into the BFS container with a BFS device, the BFS device comprising Included is a BFS dispenser that can dispense the recipe into the BFS container, and a BFS delivery system configured to deliver the recipe to the BFS dispenser, the BFS delivery system having one or more cooling features, The cooling component is configured to maintain the formulation at a low temperature (e.g., below 15 °C, such as 2-10 °C, such as 8 °C) prior to dispensing the formulation into the BFS container, wherein the chimeric cytokine The protein comprises a chimeric IL-1 (interleukin-1) family cytokine domain, wherein at least a first segment of the region is at least 20 amino acids in length and is associated with the first IL-1 The corresponding segment of the family cytokine has at least 80% amino acid identity, and at least a second segment of the region is at least 20 amino acids in length and is associated with a second IL-1 family cytokine The corresponding segment has at least 80% amino acid Identity, wherein the first and second IL-1 family cytokines are selected from the group consisting of human IL-1β (SEQ ID NO: 16), human IL-1α (SEQ ID NO: 17 ), as well as human IL-1Ra (sequence identification number: 18). The method of claim 1, wherein the chimeric cytokine region binds to IL-1R receptor I (IL-1RI) (eg, has a Kd of less than 5 nM), and Inhibition of IL-1RI activity. The method of claim 1 or 2, wherein the chimeric cytokine protein in the formulation has an unfolding temperature of at least 36 °C. A method of providing a stable formulation in a blow molded fill seal (BFS) container, the formulation comprising a chimeric cytokine protein, the method comprising dispensing the formulation into the BFS container with a BFS device, the BFS device comprising a BFS dispenser for dispensing the recipe into the BFS container, and a BFS delivery system configured to deliver the recipe to the BFS dispenser, the BFS delivery system having one or more cooling features, The cooling component is configured to maintain the formulation at a low temperature prior to dispensing the formulation into the BFS container, wherein the chimeric cytokine protein comprises a sequence, the sequence and P01 (SEQ ID NO: 1), P02 (sequence identification number: 2), P03 (sequence identification number: 3), P04 (sequence identification number: 4), or P05 (sequence identification number: 5) have at least 90% identity. The method of claim 4, wherein the chimeric cytokine protein comprises a sequence comprising P01 (SEQ ID NO: 1), P02 (SEQ ID NO: 2), P03 (SEQ ID NO: 3), P04 (Sequence Identification Number: 4), or P05 (Sequence Identification Number: 5) has at least 95% identity. The method of claim 5, wherein the chimeric cytokine protein is P05 (SEQ ID NO: 5). The method of any one of clauses 1 to 6, wherein the cooling component comprises The column is either constructed of one or more lines within the BFS delivery system that are insulated and/or cooled. The method of any of claims 1-7, wherein the cooling component comprises a heat exchanger. The method of claim 8 wherein the heat exchanger circulates the refrigerant around one or more of the lines within the BFS delivery system. The method of any of claims 1-9, further comprising monitoring the temperature of the formulation within the BFS delivery system at a location adjacent to the dispenser, such as using a thermocouple. The method of claim 10, wherein the temperature of the formulation at the location of the thermocouple is 2-10 ° C (such as 8 ° C). The method of any of claims 1-11, further comprising maintaining the temperature of the formulation in the storage tank in the BFS device at 2-8 ° C (such as 3 ° C), and wherein the BFS delivery system is The formation of the formulation from the storage tank into the dispenser. The method of any of claims 1-12, wherein the formulation is an aqueous formulation comprising a citrate buffer such as sodium citrate. The method of any of claims 1-13, wherein the formulation is an aqueous formulation comprising a sugar such as sucrose. The method of any of claims 1-14, wherein the formulation comprises sucrose, citrate, and a poloxamer, such as poloxamer 188. The method of claim 15, wherein the formulation comprises 1 to 50 mg/ml of the chimeric cytokine protein; 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 Where the formulation has a pH of 5.5 to 7.5. The method of claim 16, wherein the formulation consists of: 1 to 50 mg/ml of the chimeric cytokine protein; 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. The method of claim 17, wherein the formulation does not contain sodium carboxymethylcellulose. The method of any one of claims 1 to 18, wherein the formulation comprises P05 (SEQ ID NO: 5) of 1-20 mg/ml. The method of any one of claims 1 to 19, wherein the formulation consists of 10 mM sodium citrate, 5% sorbitol, 0.1% poloxamer 188, and 1-20 mg/ml (eg 5mg/ml) P05. A stable aqueous formulation encapsulated in a BFS container comprising 1-20 mg/ml of IL-1β/IL-1Ra chimeric cytokine protein having at least 90 with P01, P02, P03, P04, or P05 % identity, 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 5.5 To a pH of 7.5. The formulation of claim 21, wherein the chimeric cytokine protein is P05. The formulation of claim 21 or 22, wherein the BFS container is contained within a package, such as an aluminum foil pouch, optionally having an inert gas (e.g., nitrogen) overlay, optionally The encapsulation protects the formulation from photoinduced degradation and, optionally, the encapsulation and/or the blunt coating protects the formulation from oxidation. The method of any one of claims 1 to 20, or the formulation of any one of claims 21-23, wherein when evaluated using a light obscuration particle count test 10 μm particles are present with less than or equal to 50 particles per milliliter, and/or When 25 μm particles are present with less than or equal to 5 particles per ml, the formulation is stable. The method of any one of claims 1 to 20, or the formulation of any one of claims 21 to 23, wherein the protein solution is vortexed at room temperature, for example at 25 ° C for 4 hours, relative to In the form of aggregation, when the monomeric form of the protein is >90%, it indicates that the formulation is stable. The method or formulation of claim 25, wherein the percentage of the protein monomer form in the formulation relative to the aggregated form is assessed using SEC-HPLC. The method of any one of claims 1 to 20, wherein the formulation of any one of claims 21-23, wherein the formulation is stored in the BFS container at 2-8 ° C and 60% relative humidity After at least 5 months, and/or around the strip The formulation is stable, for example, at room temperature, for example at 25 ° C, after storage in the BFS container for at least 5 months. The method or formulation of claim 27, wherein the formulation is stable at at 2-8 ° C and 60% relative humidity for at least 12 months after storage in the BFS container, and/or under ambient conditions, For example, at room temperature, for example at 25 ° C, storage in the BFS container is stable for at least 6 months. The method or formulation of claim 27 or 28, wherein the formulation is stable by one or more of the following (e.g., 1, 2, 3, 4 or all): (i) using SEC- When evaluated by HPLC, the monomeric form is present at >90% (a/a), (ii) the primary band is in compliance with the reference standard in reduced SDS-PAGE, and (iii) the primary band is consistent with Reference standard in non-reduced SDS-PAGE, (iv) When using weak cation exchange HPLC (WCEX-HPLC) to evaluate the formulation, the main peak is greater than or equal to 85% (a/a), and (v) when using RP - HPLC to evaluate the formulation, the main peak is greater than or equal to 80% (a / a). A kit comprising the formulation encapsulated in a BFS container according to any one of claims 21-29, and optionally, instructions for use.