Suspension formulations containing protein particles suspended in a non-aqueous solvent

Abstract

The present invention provides a suspension formulation comprising protein particles suspended in a non-aqueous solvent, the particles comprising a protein and a stabilizer, wherein the residual water content of the suspended protein particles is less than 1.0 wt. % based on the total weight of the particles.

Description

[Background technology]

[0001] Formulations based on proteins and antibodies as active ingredients are typically formulated as aqueous solutions or lyophilized products, which require reconstitution before use and administration. However, protein instability is common in aqueous solutions, resulting in such formulations having limited shelf life and / or the development of complex cold-chain solutions. An alternative approach is to provide protein drugs as lyophilized (i.e., freeze-dried) solid powder forms, but lyophilized products require careful and precise reconstitution in an aqueous medium under sterile conditions before use, which is generally less convenient for patients and healthcare providers. The reconstitution process itself can cause aggregation if the pH or temperature of the aqueous medium is suboptimal, if the time available for rehydration is too short, or if the vial is shaken too vigorously during the dissolution process. Failure to properly dissolve a lyophilized antibody formulation within the recommended time period often necessitates discarding the sample, which increases the likelihood of waste in addition to aggregation.

[0002] The use of ready-to-use liquid formulations is generally preferred as they are easier for the user to prepare for administration. As an alternative to aqueous formulations, suspensions of proteins in non-aqueous carriers and solvents have been described.

[0003] For example, International Patent Application Publication No. 2013 / 110621 describes the formulation of protein and polypeptide preparations in semi-fluorinated alkanes. International Patent Application Publication No. 2015 / 011119 also describes antibodies suspended in semi-fluorinated alkanes as a means for formulating these types of compounds. Formulating proteins, polypeptides, and antibodies in semi-fluorinated alkanes is described as preventing degradation or aggregation of these molecules.

[0004] However, there remains a need to provide non-aqueous suspension formulations of protein particles, i.e., particles comprising a protein and one or more excipient(s), such as a stabilizer, that are suitable for storage and resistant to changes in storage conditions, such as fluctuations in elevated storage temperatures. There also exists a need to provide suspensions of protein particles comprising a protein / polypeptide and one or more excipient(s), that have stable suspension properties, e.g., particle size and particle size stability, that allow for injection of the suspension and subsequent storage.

[0005] Therefore, it is an object of the present disclosure to provide a non-aqueous protein particle suspension formulation that can be stable upon storage and injection. A further object is to provide a process or method for preparing the non-aqueous protein particle suspension formulation. Further objects of the present invention will become apparent based on the following description of the invention, examples, and claims. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Patent Application Publication No. 2013 / 110621 [Patent Document 2] International Publication No. 2015 / 011119 Summary of the Invention

[0007] In a first aspect, the present invention relates to a method for preparing a suspension formulation comprising protein particles and a non-aqueous solvent, the method comprising the steps of: a) providing an aqueous solution comprising a protein and a stabilizer; b) removing water from the aqueous solution comprising the protein and the stabilizer to obtain solid protein particles; c) further drying the protein particles obtained in step b) to obtain protein particles having a residual water content of less than 0.5 wt. % based on the total weight of the particles; and d) suspending the protein particles of step c) in a non-aqueous solvent; and optionally e) homogenizing the suspension formulation, preferably by high-shear homogenization, milling or sonication.

[0008] The present invention also relates to a composition comprising protein particles obtainable by the method of the present invention.

[0009] In a further aspect, the present invention relates to a suspension formulation comprising protein particles suspended in a non-aqueous solvent, the particles comprising a protein and a stabilizer, wherein the residual water content of the suspended protein particles is less than 0.5% by weight, based on the total weight of the particles.

[0010] In yet a further aspect, the present invention provides the use of the suspension formulations described for therapeutic and / or diagnostic applications. [Brief explanation of the drawings]

[0011] [Figure 1] Shown from left to right are particle size distributions of protein particle suspensions containing lysozyme and sucrose (50:50 ratio) in a non-aqueous solvent, such as 5a (F6H8; never-dried particles), 5b (F6H8; vacuum-dried particles), 6a (EO; never-dried particles), and 6b (EO; vacuum-dried particles), as listed in Table 1. These suspensions were prepared by homogenization in an ice-cooled ultrasonic bath.

[0012] [Figure 2]Particle size distributions of protein particle suspensions containing a model monoclonal antibody (mAb) and sucrose (50:50 ratio, 100 mg / mL total solids) in non-aqueous solvents are shown from left to right. These suspensions were prepared in an ice-cooled ultrasonic bath. These suspensions were: 7a (F4H5, wet particles), 7b (F4H5, vacuum-dried particles), 8a (F6H8, wet particles), 8b (F6H8, vacuum-dried particles), 9a (ethyl oleate, wet particles), 9b (ethyl oleate, vacuum-dried particles), 10a (medium-chain triglyceride, wet particles), and 10b (medium-chain triglyceride, vacuum-dried particles). These suspensions were prepared in an ice-cooled ultrasonic bath.

[0013] [Figure 3] Figure 1 shows particle size distributions of protein particle suspensions containing a model monoclonal antibody (mAb) and the stabilizer sucrose (mAb:Suc, 50:50, TSC = 100 mg / mL), including 11a (F6H8, wet particles), 11b (F6H8, vacuum-dried particles), 12a (MCT, wet particles), and 12b (MCT, vacuum-dried). The suspensions were homogenized using a high-shear homogenizer.

[0014] [Figure 4] Particle size distributions of protein particle suspensions containing bevacizumab (bevacizumab) and the stabilizer sucrose (bevacizumab:sucrose 50:50, TSC = 100 mg / mL), including 14a (F6H8, wet particles), 14b (F6H8, vacuum-dried particles), 15a (EO, wet particles), and 15b (EO, vacuum-dried), are shown from left to right. These suspensions were prepared in an ultrasonic bath for homogenization.

[0015] [Figure 5]The particle size distributions of the following suspension formulations are shown from left to right: suspension formulation 8a (F6H8, mAb:Suc 50:50, TSC = 100 mg / mL) prepared from protein particles that were not vacuum dried after storage at 5°C for 6 months; suspension formulation 8b (F6H8, mAb:Suc 50:50, TSC = 100 mg / mL) prepared from protein particles that were vacuum dried after storage at 5°C for 6 months; suspension formulation 8a after storage at 25°C for 6 months; and suspension formulation 8b after storage at 25°C for 6 months.

[0016] [Figure 6A] Figure 6 shows the particle size distribution of suspension formulations of protein particles containing a model mAb and sucrose in F4H5 as a liquid solvent after storage at 40°C for 0, 1, 3, and 6 months. Figure 6A shows the particle size distribution of suspension formulation 7a (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from particles that had not been subjected to vacuum drying and contained a residual water content of approximately 4.2 wt%. [Figure 6B] Figure 6B shows the particle size distribution of suspension formulations of protein particles containing a model mAb and sucrose in F4H5 as a liquid vehicle after storage at 40 °C for 0, 1, 3, and 6 months. Figure 6B shows the particle size distribution of suspension formulation 7b (50:50 mAb:Sucrose, TSC = 100 mg / mL) containing a residual water content of approximately 0.1 wt%. In these figures, the particle size distributions are expressed as D5 (filled circle ●), D10 (open circle ○), D50 (filled triangle ▼), D90 (open triangle △), and D95 ​​(filled square ■) mean particle size values ​​measured using laser diffraction.

[0017] [Figure 7A] Figure 7 shows the particle size distribution of suspension formulations of protein particles containing a model mAb and sucrose in F6H8 as a liquid solvent after storage at 40°C for 0, 1, 3, and 6 months. Figure 7A shows the particle size distribution of suspension formulation 8a (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from particles that had not been subjected to vacuum drying and contained a residual water content of approximately 4.2 wt%. [Figure 7B] Figure 7B shows the particle size distribution of a suspension formulation of protein particles containing a model mAb and sucrose in F6H8 as a liquid vehicle after storage at 40 °C for 0, 1, 3, and 6 months. Figure 7B shows the particle size distribution of suspension formulation 8b (50:50 mAb:Sucrose, TSC = 100 mg / mL) containing a residual water content of approximately 0.1 wt%. In these figures, the particle size distribution is expressed as the D5 (filled circle ●), D10 (open circle ○), D50 (filled triangle ▼), D90 (open triangle △), and D95 ​​(filled square ■) mean particle size values ​​measured using laser diffraction.

[0018] [Figure 8] 1 shows the XRD spectrum of particles of formulation 8a (F6H8, 50:50 mAb:Suc, TSC=100 mg / mL, prepared without vacuum drying) after 6 months of storage at 5°C, 25°C, and 40°C (spectrum A), and the XRD spectrum of protein particles of formulation 8b (50:50 mAb:Suc, TSC=100 mg / mL, prepared with vacuum drying) after 6 months of storage at 5°C, 25°C, and 40°C (spectrum B).

[0019] [Figure 9A] Figure 9 shows the particle size stability of formulations made with spray-dried and vacuum-dried particles containing lysozyme and trehalose and F6H8 as a liquid solvent after storage for 0, 1, 3, 6, and 12 months at 40° C. Figure 9A shows the particle size distribution of suspension formulation 2 (Lys:Tre 70:30, TSC=300 mg / mL). [Figure 9B] Figure 9B shows the particle size stability of formulations made with spray-dried and vacuum-dried particles containing lysozyme and trehalose and F6H8 as the liquid solvent after storage for 0, 1, 3, 6, and 12 months at 40° C. Figure 9B shows the particle size distribution of suspension formulation 3 (PS20 containing a 70:30 Lys:Tre formulation, TSC=100 mg / mL). [Figure 9C]Figure 9C shows the particle size stability of formulations made with spray-dried and vacuum-dried particles containing lysozyme and trehalose and F6H8 as the liquid vehicle after storage at 40°C for 0, 1, 3, 6, and 12 months. Figure 9C shows the particle size distribution of suspension formulation 4 (Lys:Tre 50:50, TSC = 100 mg / mL). In these figures, the particle size distribution is expressed as D5 (filled circle ●), D10 (open circle ○), D50 (filled triangle ▼), D90 (open triangle △), and D95 ​​(filled square ■) particle size values ​​(mean particle size) measured using laser diffraction.

[0020] [Figure 10] Shown from left to right are the resuspendability of suspension formulations 1a, 1b, 1c, and 1d (Lys:Tre 70:30, TSC = 100 mg / mL, liquid solvents F4H5, F6H8, EO, and MCT, respectively) stored at 5°C, 25°C, and 40°C for 12 months. Resuspension was determined using a vertical shaker (vertical rotation at 25 rpm) and is based on the time required for resuspension as determined by visual inspection.

[0021] [Figure 11] The resuspension properties of suspension formulations are shown from left to right for each group of formulations 5a, 5b, 6a, and 6b (Lys:Suc 50:50, TSC=100 mg / mL, as described in Table 1: 5a (F6H8, wet particles), 5b (F6H8, vacuum-dried), 6a (EO, wet particles), and 6b (EO, vacuum-dried)). Figure 11A shows the resuspension properties based on the time required for resuspension based on vertical rotation, as determined by visual inspection, and Figure 11B shows the resuspension properties based on the shaking method, i.e., the shaking frequency required for resuspension. In Figure 11B, the horizontal line marked at 5 Hz represents the frequency used by an average person for this procedure.

[0022] [Figure 12]The resuspension properties of suspension formulations 7a, 7b, 8a, 8b, 9b, and 10b (50:50 mAb:Suc, TSC=100 mg / mL) listed in Table 1 are shown from left to right after storage at 40°C for 1 month, 40°C for 3 months, or 5°C, 25°C, and 40°C for 6 months. The resuspension properties are based on the shaking frequency required for resuspension. In B, the horizontal line marked at 5 Hz depicts the frequency used by an average human for this procedure.

[0023] [Figure 13A] Figure 13A shows the maximum injection force (or gliding force) required for injection using a 27G needle and a 1 mL syringe to achieve a volumetric flow rate of 0.1 mL / sec for Formulations 1a, 1b, 1c, and 1d, which are suspension formulations containing lysozyme-trehalose-containing particles (lysozyme:trehalose 70:30, TSC = 100 mg / mL) as described in Table 1, over a 12-month storage period at 40°C. Figure 13A shows the results of the injectability test for Formulation 1a (solvent F4H5) and Formulation 1b (solvent F6H8). [Figure 13B] Figure 13B shows the maximum injection force (or gliding force) required for injection to achieve a volumetric flow rate of 0.1 mL / sec using a 27 G needle and a 1 mL syringe for Formulations 1a, 1b, 1c, and 1d, which are suspension formulations containing lysozyme-trehalose-containing particles (lysozyme:trehalose 70:30, TSC = 100 mg / mL), as described in Table 1, over a 12-month storage period at 40°C. Figure 13B shows the results of an injectability test for Formulation 1c (solvent EO) and Solvent 1d (solvent MCT).

[0024] [Figure 14] Figure 1 shows the gliding force profiles of Formulation 2 (top curve) and Formulation 3 (bottom curve) after 12 months of storage at 40°C, based on the force required for injection using a 27G needle and a 1 mL syringe at a volumetric flow rate of 0.1 mL / sec.

[0025] [Figure 15]Figure 15A shows the maximum injection force (or gliding force) required for injection using a 27G needle and a 1 mL syringe to achieve a volumetric flow rate of 0.1 mL / sec for suspension formulations 7a and 7b (F4H5, 50:50 mAb:Suc, TSC = 100 mg / mL) and 8a and 8b (F6H8, 50:50 mAb:Suc, TSC = 100 mg / mL) containing model mAb-sucrose-containing protein particles, as described in Table 1, stored at 40°C for 6 months. Figure 15A shows the results of a syringeability test for formulations 7a and 8a, which were not subjected to an additional vacuum drying step and were prepared with protein particles having a residual water content of approximately 4.2% by weight. Figure 15B shows the results for suspension formulations 7b and 8b, which were prepared with protein particles that were dried under vacuum after spray drying and had a residual water content of approximately 0.1% by weight, based on the particle weight.

[0026] [Figure 16] Figure 1 shows the gliding force profiles of suspension formulation 10b (top curve) and suspension formulation 9b (bottom curve) in Table 1 after 6 months of storage at 40°C, based on the force required for injection using a 27G needle and a 1 mL syringe at a volumetric flow rate of 0.1 mL / sec.

[0027] [Figure 17] The graph shows the maximum injection force (or gliding force) required to achieve a volumetric flow rate of 0.1 mL / sec using a 27G needle and a 1 mL syringe for suspension formulations 14a and 14b (F6H8, beva:suc 50:50, TSC 100 mg / mL) with bevacizumab-sucrose-containing protein particles after 3 months of storage at 40° C. (14a, 14b) and for suspension formulations 15a and 15b (EO, beva:suc 50:50, TSC 100 mg / mL) with bevacizumab-sucrose-containing protein particles after 6 months of storage at 40° C. (14a, 14b, 15a, 15b). From left to right, the measurements for 14a, 14b, 14a, 14b, 15a, and 15b are shown. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present inventors have surprisingly found that compositions comprising protein particles comprising a protein and a stabilizer, wherein the protein particles are suspended in a non-aqueous solvent, exhibit highly advantageous properties when the protein particles are prepared according to the methods of the present invention.

[0029] In particular, compositions containing protein particles obtained by two different successive drying processes provide chemical and physical stability, and an improved particle size distribution combined with easy redispersibility allows for improved injection processing through a syringe.

[0030] Thus, in a first aspect, the present invention relates to a method for preparing a suspension formulation comprising protein particles and a non-aqueous solvent, the method comprising: a) providing an aqueous solution comprising a protein and a stabilizing agent; b) removing water from the aqueous solution containing the protein and the stabilizer to obtain solid protein particles; c) further drying the protein particles obtained in step b) to obtain protein particles having a residual moisture content of less than 0.5 wt. %, based on the total weight of the particles; d) suspending the protein particles of step b) in a non-aqueous solvent, and optionally e) homogenizing the suspension formulation, preferably by high shear homogenization, milling or sonication; The protein particles include a protein and a stabilizer, and the non-aqueous solvent includes a semi-fluorinated alkane, a medium chain triglyceride (MCT), ethyl lactate, ethyl oleate, or a mixture thereof.

[0031] The method of the present invention is suitable for producing a protein particle suspension with surprisingly improved properties. This method essentially comprises two drying steps. The first step (b) involves removing water from an aqueous solution containing a protein and a stabilizer to obtain protein particles, resulting in a residual water content of less than 5% by weight, less than 3% by weight, or in the range of 3-5% by weight. The second step reduces the residual water content of the obtained protein particles to less than 0.5% by weight. The obtained protein particle suspension is characterized by excellent chemical and physical stability, easy redispersibility, and a favorable particle size distribution. The particle size distribution of the protein particle suspension obtained by this method is ideal for injection purposes, preventing clogging of needles or cannulas.

[0032] Thus, in a preferred embodiment, step b) of removing water from the aqueous solution comprising the protein and the stabilizer is carried out using a highly effective drying method, preferably a drying process resulting in a residual moisture content of the protein particles of less than 5% by weight, or less than 3% by weight, or a drying method resulting in a residual moisture content of the protein particles in the range of 3-5% by weight, or in the range of 1-3% by weight. Suitable methods are known to those skilled in the art. Examples of such methods include lyophilization (i.e., freeze-drying) or spray-drying.

[0033] Thus, in one embodiment, step b) comprises spray drying or lyophilizing (or freeze-drying) an aqueous solution comprising the protein and the stabilizing agent to obtain solid protein particles.

[0034] The second drying step may be carried out by any, possibly other suitable, drying method capable of (further) reducing the residual moisture content of the protein particles to less than 0.5 wt. % based on the total weight of the particles. A preferred method is vacuum drying.

[0035] In one embodiment, the suspension formulation may be obtained by step b) comprising spray-drying an aqueous solution comprising the protein and a stabilizer, and optionally further excipients (e.g., a buffer such as histidine), to obtain protein particles. In another embodiment, the suspension formulation may be obtained by step b) comprising lyophilization (i.e., freeze-drying) of an aqueous solution comprising the protein and a stabilizer, and optionally further excipients (e.g., a buffer such as histidine), to obtain protein particles.

[0036] In step b), spray drying may be carried out using, for example, but not limited to, a cyclone spray dryer. The inlet / outlet temperatures of the spray dryer used in this method should be such that they do not adversely affect protein loss or degradation. In one embodiment, the spray drying process temperature does not exceed 130°C. In another embodiment, the spray drying process does not exceed 130°C, i.e., 130°C or less (inlet temperature) and 70°C or less (outlet temperature).

[0037] In a preferred embodiment, step c) comprises vacuum-drying the particles of step b). In step c), the vacuum drying may be carried out at ambient temperature or at a temperature slightly above ambient temperature, for example, between 15°C and 40°C. In some embodiments, the vacuum drying may be carried out at a temperature of 20°C to 35°C, or 22°C to 35°C, or 25°C to 33°C, or 27°C to 32°C. The vacuum drying is preferably carried out under reduced pressure, for example, between 0.01 and 100 mbar. In other embodiments, the vacuum drying may be carried out at 0.01 to 10 mbar, 0.01 to 1 mbar, or 0.01 mbar. In one embodiment, the vacuum drying of the particles obtained from step b) in step c) may be carried out at 15 to 40°C and a pressure of 0.01 to 100 mbar. The duration of the vacuum drying in step b) may be at least 6 hours, or at least 12 hours, or at least 24 hours. In one embodiment, step c) is carried out at a temperature of 15-40°C and a pressure of about 0.01-100 mbar for a period of at least 24 hours. In an alternative embodiment, vacuum drying step c) may be carried out for 24 hours or less. Step c) may be carried out to obtain protein particles having a residual moisture content of less than 0.5 wt. %, based on the total weight of the particles.

[0038] All compounds utilized in this method may be dried or water-free. The resulting suspension formulation is essentially water-free or substantially water-free. In some embodiments of the present invention, the residual water content of the suspension formulation is less than 0.5 mg / mL (or less than 0.05% (v / v)), based on the total volume of the formulation.

[0039] In step d), the protein particles are suspended in a non-aqueous liquid solvent comprising a semi-fluorinated alkene, a medium chain triglyceride (MCT), ethyl lactate, ethyl oleate, or a mixture thereof.

[0040] Preferably, in step d), the protein particles are suspended in a non-aqueous liquid solvent comprising a semi-fluorinated alkene. Semi-fluorinated alkanes are substantially non-toxic and have been found to be well tolerated by a wide variety of human and animal tissues when administered topically or parenterally. In addition, they are chemically inert and generally compatible with active and inactive ingredients in pharmaceutical formulations. Their typical density is 1.1-1.7 g / cm. 3 and these surface tensions can be as low as 19 mN / m.

[0041] Semi-fluorinated alkanes are linear or branched alkanes in which some hydrogen atoms have been replaced by fluorine atoms. In one embodiment, the semi-fluorinated alkanes (which may be abbreviated as SFA) described and used in this disclosure are composed of one linear non-fluorinated hydrocarbon segment and one linear perfluorinated hydrocarbon segment, preferably with the perfluorinated hydrocarbon segment bonded to the non-fluorinated hydrocarbon segment.

[0042] In one embodiment, the semi-fluorinated alkane has the formula F(CF2) n (CH2) m where n and m are integers defining the number of carbon atoms in the perfluorinated hydrocarbon segment and the number of carbon atoms in the non-fluorinated hydrocarbon segment, respectively. In further embodiments, the one or more semi-fluorinated alkanes have the formula F(CF2) n (CH2) m where n is an integer selected from 4 to 6 and m is an integer selected from 2 to 10. In a further embodiment, the one or more semi-fluorinated alkanes have the formula F(CF2) n (CH2) m wherein n is an integer selected from 4 to 6, and m is an integer selected from 4 to 8.

[0043] The name frequently used for semi-fluorinated alkanes indicates the perfluorohydrocarbon segment as RF and the non-fluorinated segment as RH. Alternatively, the compounds may be referred to as FnHm and FnHm, respectively, where F refers to the perfluorohydrocarbon segment, H refers to the non-fluorinated segment, and n and m define the number of carbon atoms in each segment. For example, F3H3 is used for perfluoropropylpropane (F(CF2)3(CH2)3H). Furthermore, this type of name is usually used for compounds with linear chains (i.e., unbranched segments). Therefore, unless otherwise indicated, F3H3 should be assumed to refer to 1-perfluoropropylpropane, not 2-perfluoropropylpropane, 1-perfluoroisopropylpropane, or 2-perfluoroisopropylpropane.

[0044] Semi-fluorinated alkanes of the RFRH type are insoluble in water but also somewhat amphiphilic, with the increase in lipophilicity being associated with an increase in the size of the non-fluorinated segments. The semi-fluorinated alkanes used in the context of the present disclosure are preferably liquid semi-fluorinated alkanes.

[0045] In one embodiment of the present disclosure, the non-aqueous solvent may be comprised of one or more semi-fluorinated alkanes selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H2, F6H4, F6H6, F6H8, and F6H10; or the non-aqueous solvent may be comprised of one or more fluorinated alkanes selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and F8H8; or the non-aqueous solvent may be comprised of one or more semi-fluorinated alkanes selected from the group consisting of F4H5, F4H6, F4H8, F6H6, and F6H8. The chemical formulas of these semi-fluorinated alkanes are F(CF2)4(CH2)4H, F(CF2)4(CH2)5H, F(CF2)4(CH2)6H, F(CF2)4(CH2)8H, F(CF2)6(CH2)2H, F(CF2)6(CH2)4H, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H and F(CF2)6(CH2), respectively. 10H. In another embodiment, the non-aqueous solvent consists essentially of one or more semi-fluorinated alkanes selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H4, F6H6, and F6H8.

[0046] In one embodiment, a suspension formulation according to the present disclosure comprises protein particles, such as those defined herein, suspended in a non-aqueous solvent comprising or consisting essentially of F6H8. In another embodiment, a suspension formulation according to the present disclosure comprises protein particles, such as those defined herein, suspended in a non-aqueous solvent comprising or consisting essentially of F4H5. In another embodiment, a suspension formulation according to the present disclosure comprises protein particles, such as those defined herein, suspended in a non-aqueous solvent selected from F4H5 and F6H8.

[0047] Optionally, the formulation may contain two or more semi-fluorinated alkanes. For example, it may be useful to combine SFAs to achieve a particular desired property, such as a particular density or viscosity. When a mixture of semi-fluorinated alkanes is used, the mixture preferably contains at least one of F4H4, F4H5, F4H6, F4H8, F6H4, F6H6, and F6H8, or at least one of F4H4, F4H5, F4H6, F4H8, F6H2, F6H4, F6H6, F6H8, and F6H10. In one embodiment, the non-aqueous solvent may comprise at least two members selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H4, F6H6, and F6H8, or may comprise at least two members selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H2, F6H4, F6H6, F6H8, and F6H10.

[0048] As used herein, a non-aqueous solvent comprising a semi-fluorinated alkane comprises at least one or more semi-fluorinated alkanes. The solvent may optionally further comprise other solvents or simple compounds, or excipients, as further described herein. In one embodiment, the liquid solvent comprises two or more semi-fluorinated alkanes. In another embodiment, the liquid solvent consists essentially of a semi-fluorinated alkane or a mixture of semi-fluorinated alkanes, such as any one of the semi-fluorinated alkanes defined herein. In another embodiment, a suspension formulation comprises a non-aqueous solvent consisting of one or more semi-fluorinated alkanes and, optionally, one or more pharmaceutically acceptable excipients, preferably those that are miscible or soluble in the semi-fluorinated alkane or semi-fluorinated alkane mixture. In one embodiment, the non-aqueous solvent comprises the semi-fluorinated alkane or mixture of semi-fluorinated alkanes in an amount of at least 70%, 75%, 85%, 90%, 95%, or at least 99% by weight, based on the total weight of the liquid solvent. In a further embodiment, the non-aqueous solvent consists essentially of 100% by weight of the semi-fluorinated alkane or mixture of semi-fluorinated alkanes, as defined above.

[0049] In another embodiment, the method may include step e) homogenizing the suspension formulation. Homogenization may be performed by any homogenization technique known in the art, such as using a high-shear homogenizer or by ultrasound, optionally under cooled conditions (e.g., under ice-cold conditions, such as at about 0°C).

[0050] In a preferred embodiment, the method comprises step e), wherein the homogenization is carried out using sonication. In a further embodiment, the sonication is carried out below ambient temperature, preferably under chilled conditions, such as under ice-cold conditions (in an ice bath).

[0051] In another embodiment, the method may include an optional step of selecting protein particles having a desired or predetermined particle size, where the particle size is defined by an average particle size. Preferably, the selection of protein particles having a desired or predetermined particle size may be performed before suspending the protein particles in the non-aqueous solvent. The selection of protein particles having a desired or predetermined particle size may be performed by any method known to those skilled in the art, and may include an additional grinding step to generate or increase the number of particles having a smaller desired or predetermined particle size, and / or may include a step of sorting (i.e., by picking or sieving) the suspended particles having the desired or predetermined particle size. The desired or predetermined particle size is defined by the intended use or medical application of the suspension formulation. For example, when utilized for injection purposes such as subcutaneous, intramuscular, or intraocular injection, the predetermined particle size may be characterized by at least a 90% distribution of particles having an average particle size of 1-15 μm, or 1-30 μm, or 1-50 μm, or the predetermined particle size may be characterized by an average particle size of less than 50 μm, less than 30 μm, less than 15 μm, 1-15 μm, 1-30 μm, or 1-50 μm, respectively, as determined by laser diffraction. In further embodiments, the aqueous solution comprises a protein and a stabilizer, wherein the relative weight ratio of protein to stabilizer is 1:1 to 7:3.

[0052] This method is suitable for obtaining protein particle suspensions of all types of proteins, including naturally occurring proteins and artificially produced proteins. In some embodiments, the protein in the aqueous solution is selected from antigen-binding polypeptides or proteins, vaccines, and enzymes. In some embodiments, the protein is an antibody or antibody fragment.

[0053] In some embodiments, the protein has a molecular mass between 10 and 300 kDa.

[0054] This method is compatible with a wide range of stabilizing agents in suspension. Examples of stabilizing agents include, but are not limited to, sugars, polyols, amino acids, amines, surfactants, antioxidants, polymers, salts, or combinations thereof. In some embodiments, the stabilizing agent is selected from sugars, polyols, amino acids, amines, surfactants, antioxidants, polymers, salts, or combinations thereof. In some embodiments, the stabilizing agent is a sugar, preferably selected from trehalose or sucrose.

[0055] The main advantage of this method is that the size distribution of the protein particles in the suspension is very uniform and the particles are small. In one embodiment, this method produces a suspension formulation comprising protein particles, at least 90% of which have an average particle size of 1-30 μm, or 1-50 μm, as determined by laser diffraction.

[0056] The protein concentration in the suspension formulation can be adjusted in step d) to suit specific needs. In some embodiments, the protein is suspended in a sufficient amount of liquid solvent such that the protein concentration in the suspension formulation is 2-350 mg / mL, 2-250 mg / mL, or 2-125 mg / mL. In some embodiments, the total solids content of the suspension formulation is 4-700 mg / mL, 7-500 mg / mL, or 4-250 mg / mL.

[0057] The protein particles may contain additional compounds in addition to the protein and the stabilizer. In some embodiments, the protein particles or the liquid solvent may additionally contain a surfactant. In a preferred embodiment, the suspension formulation obtained by this method does not contain a surfactant.

[0058] In a related aspect, the present disclosure may relate to a suspension formulation obtainable or obtainable according to any one of the above method embodiments.

[0059] In a further aspect, the present disclosure relates to a suspension formulation comprising protein particles suspended in a non-aqueous solvent and twice dried, the particles comprising a protein and a stabilizer, wherein the residual water content of the suspended protein particles is less than 0.5 wt. % based on the total weight of the particles. Herein, the present disclosure relates to a suspension formulation comprising protein particles suspended in a non-aqueous solvent and twice dried, the particles comprising a protein and a stabilizer, wherein the residual water content of the suspended protein particles has been reduced to less than 0.5 wt. % based on the total weight of the particles (from the initial aqueous solution) by two successive drying steps.

[0060] The suspension formulation can be obtained using the above method. Any particular embodiment of the suspension formulation described herein may be applied or realized by the above method.

[0061] In the context of the present invention, "double-dried protein particles" refer to solid protein particles obtained by drying a protein composition by two different methods. Preferably, the two different drying methods are performed sequentially. Preferably, double-dried particles are obtained by first drying an aqueous solution containing a protein and a stabilizer by a drying method that is highly effective for obtaining solid protein particles, such as spray drying or lyophilization (i.e., freeze-drying), followed by a (sequential) subsequent drying step, such as vacuum drying. Herein, the first drying produces protein particles characterized by a residual moisture content in the range of 3-5 wt. %, and the second drying step further reduces the residual moisture content to less than 0.5 wt. %, based on the total weight of the particles.

[0062] The protein or protein particle formulations described herein are provided in the form of a suspension. A suspension may be defined as a type of dispersion system, i.e., a system having at least one continuous (or coherent) phase and at least one discontinuous (or internal) phase dispersed in the continuous phase. In a suspension, the dispersed phase is essentially present in a solid state. In one embodiment of the present disclosure, the protein particles are insoluble in the continuous phase, which is composed of a non-aqueous liquid solvent and is characterized as the dispersed phase in a suspension formulation. In a preferred embodiment, a suspension formulation according to the present disclosure is an aqueous suspension at least at physiological temperature, meaning that the continuous phase is liquid. Typically, the suspension is also liquid at room temperature.

[0063] As used and defined herein, a non-aqueous solvent can form the continuous phase of a suspension formulation. A non-aqueous solvent is preferably liquid at room temperature. As understood herein, the term "non-aqueous" in reference to a solvent or any formulation component refers to a solvent or formulation component that is essentially free of water. In another embodiment, a non-aqueous solvent is liquid and immiscible with water. As used herein, the term "solvent" may refer to a solvent that consists essentially of a single component or compound that forms the continuous phase of a suspension formulation. Alternatively, it may refer to a solvent that includes a combination of two or more components or compounds, which are preferably miscible and form a single continuous phase of a suspension formulation.

[0064] In one embodiment, the suspension formulation comprises protein particles as defined in accordance with the present disclosure suspended in a non-aqueous solvent, wherein the non-aqueous solvent comprises a semi-fluorinated alkane, a medium chain triglyceride (MCT), ethyl lactate, ethyl oleate, or a mixture thereof. In an alternative embodiment, the non-aqueous solvent is selected from the group consisting of a semi-fluorinated alkane, a medium chain triglyceride (MCT), ethyl lactate, ethyl oleate, and a mixture thereof.

[0065] In one embodiment, the non-aqueous solvent comprises one or more semi-fluorinated alkanes.

[0066] As understood herein, the phrases "essentially consists of" or "essentially consisting of" and "consists of" or "consisting of" are considered interchangeable and mean that no additional components are featured in the composition or formulation other than those listed. If any other components or ingredients, such as impurities inherent in the material, are present in the composition or formulation, they may be present in negligible or insignificant amounts and do not provide any technical contribution, advantage, or function with respect to the disclosed composition or formulation. The terms "comprises" or "comprising," in contrast, as used herein, should be construed in an open-ended sense, such that other features, such as composition ingredients, may be present apart from those prefaced by the term.

[0067] Furthermore, as used herein, the terms "about," "substantially," "essentially," and the like in connection with an attribute or numerical value, such as a concentration or amount, include the exact attribute or exact numerical value, as well as any attribute or value that is typically considered to be within a standard range or acceptable variation associated with the art and the method of measuring or determining that attribute or value.

[0068] In one embodiment, the suspension formulation comprises a liquid solvent comprising one or more semi-fluorinated alkanes, wherein the semi-fluorinated alkanes are present in an amount of at least 70%, 85%, 90%, or at least 95% by weight of the total weight of the formulation. In another embodiment, the semi-fluorinated alkanes may be present in an amount of about 85% or about 99% by weight of the formulation.

[0069] The term "protein particles" as understood herein refers to solid particles consisting of a protein that is substantially insoluble in the non-aqueous solvent of a suspension formulation, and therefore characterized as particles dispersed or suspended in a continuous phase formed by the solvent. Particles as defined herein may be composed of a protein, a stabilizer, and optionally one or more additional excipients that are combined together according to a particle preparation process, such as those further defined herein, to form a unitary solid phase that can be dispersed or suspended in a liquid, non-aqueous solvent.

[0070] As used herein, the singular forms "a," "an," or "the" do not exclude the plural; that is, these terms can be understood in addition to the singular and are meant to include the plural or plural, unless the context clearly indicates, requires, or implies otherwise. In other words, reference to individual features or limitations of the present disclosure can include the corresponding plural features or limitations, and vice versa, unless the referenced context explicitly dictates otherwise or clearly implies a contrary concept. As an example, the use of the terms "a," "an," or "the," such as in reference to "one" protein particle, has the same meaning as "at least one" or "one or more," unless otherwise defined.

[0071] As used herein, the term "protein" may be interchangeable with the term "polypeptide." A polypeptide may also be referred to as a protein, and vice versa. Typically, the term "polypeptide" refers only to a single polymer chain, while the expression "protein" can also refer to two or more polypeptides non-covalently linked to one another. In general, polypeptides and proteins refer to polymers of amino acid units linked to one another by peptide bonds. The size boundaries often used to distinguish between polypeptides and proteins are somewhat arbitrary, and the two descriptions of these molecules should not be understood to be mutually exclusive within the context of this disclosure.

[0072] In one embodiment of the present disclosure, the protein particles suspended in the non-aqueous solvent are proteins having a molecular mass of 3 to 200 kDa or 10 to 200 kDa, or 50 to 150 kDa, or 100 to 200 kDa, or 50 to 100 kDa, or 3 to 50 kDa, or 3 to 25 kDa.

[0073] In a further embodiment of the present disclosure, the protein particles suspended in the non-aqueous solvent comprise a protein, the protein being a polypeptide comprising about 25-200 amino acids, preferably about 25-100 amino acids, or more preferably about 25-50 amino acids.

[0074] In one embodiment, the protein particle may comprise an antigen-binding polypeptide or protein. As used within the context of the present disclosure, the term antigen-binding polypeptide or protein refers to full-length and complete antibodies, also known as immunoglobulins, in their monomeric or polymeric form, as well as any fragments, chains, domains, or any modifications derived from full-length antibodies that are capable of specifically binding to an antigen. The antigen-binding polypeptide or protein may belong to any of the IgG, IgA, IgD, IgE, or IgM immunoglobulin isotypes or classes. In one embodiment, the protein used herein may be an immunoglobulin G (IgG) antibody, i.e., a protein comprising an antibody, antibody fragment, or antibody fragment derived from immunoglobulin G or any of its five classes (e.g., IgG1, IgG2, IgG3, IgG4).

[0075] In one embodiment, the protein particle may include an enzyme, such as lysozyme. Lysozyme is a glycoside hydrolase enzyme that hydrolyzes glycosidic bonds such as those found in peptidoglycan. Lysozyme can function as an antibacterial agent, particularly against Gram-positive bacteria and bacteria in which peptidoglycan is a prominent feature of the bacterial cell wall. Lysozyme (chicken type) has a molecular mass of approximately 14.3 kDa. In another embodiment, the enzyme may be one that is deficient or produced in low amounts in a subject in need thereof.

[0076] In one embodiment, the protein particle may comprise a protein vaccine, such as a purified or recombinant proteinaceous antigen derived from a pathogen such as a bacterium or virus.

[0077] In another embodiment, the protein particle may comprise a protein selected from an enzyme and an antigen-binding polypeptide or protein, such as an antigen or immunoglobulin (e.g., an immunoglobulin G antibody, preferably human IgG1 or humanized IgG1), or an antigen-binding antibody fragment, or a fusion protein comprising the antibody fragment, or an antibody-drug conjugate. In yet a further embodiment, the protein is selected from the group consisting of lysozyme and an antibody or immunoglobulin.

[0078] In certain embodiments, the protein particle comprises an antibody. The term "antibody" may refer to a full-length antibody and any fragment, chain, domain, or any modification derived from a full-length antibody that is capable of specifically binding to an antigen.

[0079] Full-length antibodies are Y-shaped glycoproteins with a common structure having an Fc (fragment crystallizable) domain and an Fab (fragment antigen-binding) domain. They are structurally composed of two heavy (H) and two light (L) polypeptide chains interconnected by disulfide bonds to form a Y-shaped structure. Each type of chain contains a variable region (V) and a constant region (C), with the heavy chains containing the variable region (V) and the constant region (C). H ) and various constant regions (e.g., C H 1. C H 2), and the light chain contains a variable chain region (V L ) and constant region (C L ). The V region can be further characterized into further subdomains / regions, namely framework (FR) regions containing many conserved amino acid residues, and regions of increased variability in terms of amino acid residues, such as hypervariable (HV) regions or complementarity-determining regions (CDRs). The variable regions of the chains determine the binding specificity of the antibody and form the antigen-binding Fab domain of the antibody.

[0080] As used herein, an antibody fragment may comprise any region, chain, domain, or any construct of an antibody, or conjugate thereof, that is capable of interacting with and specifically binding to an antigen, and may be monovalent, bivalent, or even multivalent in terms of binding ability. Such antibody fragments may be produced by methods known in the art, such as, for example, dissection (e.g., proteolysis) of a full-length natural antibody, protein synthesis, genetic engineering / recombinant DNA processes, chemical cross-linking, or any combination thereof. Antibody fragments generally derive from a combination of the various domains or regions that characterize the variable V regions of a full-length antibody. In one embodiment, the protein particle may comprise an antibody fragment. For example, the antibody fragment may be an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a single-domain antibody, a minibody, or a diabody. The fragment may comprise a heavy chain variable (V) fragment joined by a linker or a hybrid multimeric / multivalent construct thereof, such as, for example, a diabody (bivalent dimer), a triabody (trivalent trimer), or a tetrabody (tetravalent tetramer). H ) domain and light chain variable (V L The antibody fragment may be a single-chain variable fragment (scFv), such as one composed of two V(I) domains. Multimeric antibody fragments may also be multispecific. For example, a bispecific diabody may be composed of two fragments, each with specificity for a different antigen. Further preferred antibody fragments include those composed of a single V(I) domain capable of specifically binding to an antigen. H Domain or V L Antibody fragments that are also within the scope of the present disclosure include scFv-C, such as minibodies, and the like. H Dimeric constructs may be mentioned.

[0081] In one embodiment, the protein contained in a particle according to the present disclosure is a monoclonal antibody (mAb). A monoclonal antibody refers to an antibody obtained from a homogeneous population of antibodies that is specific for a single epitope or binding site on an antigen. Monoclonal antibodies may be produced using antibody engineering techniques known in the art, such as by hybridomas or by recombinant DNA technology. In one embodiment, a protein particle according to the present disclosure comprises a recombinant monoclonal antibody. In further embodiments, the protein used herein may be selected from a chimeric monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. A chimeric monoclonal antibody, for example, refers to a hybrid monoclonal antibody that includes heavy or light chain domains or regions derived from antibody sequences from two or more species, such as murine and human antibody sequences. A humanized monoclonal antibody may refer to an antibody that is predominantly structurally derived from human antibody sequences, typically with at least 85-95% human sequence contribution. The term "human," on the other hand, may refer to an antibody derived exclusively from human germline antibody sequences. In one embodiment, the protein according to the present disclosure is a recombinant humanized or recombinant human monoclonal antibody, preferably an immunoglobulin G or immunoglobulin G1 antibody.

[0082] In another embodiment, the protein particle may comprise a fusion protein. A fusion protein as defined herein is composed of at least one antibody fragment capable of specifically binding to an antigen, linked to at least one other biologically active protein or polypeptide, or fragment thereof. In one embodiment, the protein particle as defined herein may be composed of an Fc fusion protein, a protein composed of an immunoglobulin Fc domain covalently linked to at least another peptide or peptide fragment.

[0083] Antibody-drug conjugates, which comprise an antibody or antibody fragment covalently bound or linked to a drug molecule (e.g., a small molecule drug, or a radiolabeled moiety), are also within the definition of antigen-binding polypeptide or protein as used herein.

[0084] In a specific embodiment, the protein is selected from bevacizumab, aflibercept, and ziv-aflibercept. Aflibercept (trade name EYLEA) is a recombinant Fc-fusion polypeptide carrying the extracellular domain of VEGF receptors (VEGF1 and VEGF2), which is used as a decoy receptor to neutralize VEGF. Aflibercept can be used to treat patients with neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), and diabetic retinopathy (DR). Bevacizumab is a recombinant humanized monoclonal antibody that blocks angiogenesis by inhibiting vascular endothelial growth factor A (VEGF-A). Bevacizumab is a full-length IgG1κ isotype antibody composed of two identical light chains and two heavy chains with a total molecular weight of 149 kDa. The two heavy chains are covalently linked to each other by two interchain disulfide bonds, which is consistent with the structure of human IgG1.

[0085] As defined herein, protein particles according to the present disclosure comprise a protein and a stabilizer. The "stabilizer" referred to herein may be any excipient or a combination of two or more excipients that stabilize the protein, protein particles, or suspension formulation itself. The stabilizer can provide protection against mechanical stress, physical stress, chemical stress, or a combination thereof during the manufacturing process or storage. For example, the stabilizer can be useful in preventing protein instability during spray drying and exposure to extreme temperatures, such as high temperatures. Examples of stabilizers include, but are not limited to, sugars, polyols, amino acids, amines, surfactants, antioxidants, polymers, salts, or combinations thereof.

[0086] In one embodiment, the stabilizer is a sugar. The sugar may be a monosaccharide, disaccharide, trisaccharide, or optionally an oligosaccharide or polysaccharide. Examples of sugars that can function as stabilizers include glucose, fructose, galactose, sucrose, maltose, trehalose, maltose, lactulose, lactose, or cyclodextrin. In a preferred embodiment, the stabilizer is selected from trehalose, sucrose, or a combination thereof. The sugar featured in or used in the production of protein particles is amorphous in one embodiment.

[0087] In another embodiment, the stabilizer is a polyol. Examples of polyols include, but are not limited to, sugar alcohols such as glycerol, arabitol, erythritol, mannitol, sorbitol, xylitol, maltitol, lactitol, etc. In yet another embodiment, the stabilizer is a sugar, a polyol, a polysorbate, or a combination thereof.

[0088] As used herein, the term "excipient" refers to any pharmaceutically acceptable agent or combination of agents that can be added to a pharmaceutical formulation or composition in an amount to provide or adjust a particular property or characteristic of the formulation or composition. Examples of excipients include surfactants, chelating agents, buffers, pH adjusters such as inorganic or organic salts, antioxidants or reducing agents, bulking agents, organic cosolvents, and stabilizers as described above. An excipient may serve or have more than one function in a formulation. The excipients used herein are preferably pharmaceutically acceptable. This means that the compound or mixture used as an excipient is non-toxic and acceptable for human pharmaceutical use. Preferably, the excipient is suitable for parenteral, topical, dermatological, or ophthalmic use.

[0089] Examples of surfactants include nonionic surfactants and ionic surfactants. Examples of surfactants that can be used in the context of the present disclosure include, but are not limited to, polysorbates and poloxamers. Poloxamers are triblock copolymers of polyoxyethylene and polyoxypropylene, and examples include poloxamer P188. Polysorbates are PEGylated sorbitan fatty acid esters, and examples that may be useful in accordance with the present disclosure include, but are not limited to, polysorbate 20 (polyoxyethylene sorbitan monolaurate), polysorbate 40 (polyoxyethylene sorbitan monopalmitate), polysorbate 60 (polyoxyethylene monostearate), and polysorbate 80 (polyoxyethylene monooleate). In one embodiment, the suspension formulation includes a surfactant. In a further embodiment, the suspension formulation does not include a surfactant.

[0090] Examples of chelating agents that can be used in the context of the present disclosure include EDTA and citrate. Examples of antioxidants include α-tocopherol, butylated hydroxytoluene, ascorbic acid, cysteine, and methionine. Examples of inorganic salts include calcium salts, magnesium salts, zinc salts, or sodium salts, such as carbonates (e.g., calcium carbonate), hydroxides, phosphates, hydrogen phosphates, acetates, or chlorides (e.g., NaCl). Examples of amino acids include arginine, histidine, glycine, glutamine, asparagine, and the like. Examples of polymers include polyvinylpyrrolidone, cellulose, and polysaccharides.

[0091] In one embodiment, the formulation includes a buffer that controls changes in pH, such as a buffer that controls pH in the range of 5.0 to 7.0, or at a pH of about 6.0. Examples of buffers include, but are not limited to, histidine, glycine, acetate, citrate, Tris, glutamate, and phosphate.

[0092] In one embodiment, the protein particles may comprise a protein, a stabilizer, and optionally one or more additional excipients. In such an embodiment, the stabilizer is different from the one or more additional excipients. In one embodiment, the protein particles may further comprise, or consist of, a protein, a stabilizer, and a buffer. In such an embodiment, the buffer may be histidine. In another embodiment, the protein particles may further comprise, or consist of, a protein, a stabilizer, a buffer (e.g., histidine), and a surfactant (e.g., polysorbate). In other embodiments, the protein particles may consist essentially of a protein, a stabilizer, and optionally one or more additional excipients.

[0093] In the protein particle, the relative weight ratio of protein to stabilizer may be in the range of 1:1 to 7:3. In other embodiments, the relative weight ratio of protein to stabilizer may be about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, or in the range of 55:45 to 70:30, 60:40 to 70:30, or 65:35 to 70:30. In a specific embodiment, the protein particle comprises an antibody or an enzyme, and the stabilizer is a sugar (e.g., trehalose or sucrose), and the relative weight ratio thereof in the protein particle is 1:1 to 7:3.

[0094] According to the present disclosure, the residual water content of the suspended protein particles is less than 0.5% by weight, based on the total weight of the particles. As understood herein, the term "water content" or "residual water content" refers to the amount of water present in a composition (e.g., protein particles) or the amount of water remaining in a composition, such as after processing or manufacturing of the composition, which may include a water removal step.

[0095] In one embodiment, the residual moisture content of the suspended particles is less than 0.5 wt%, based on the total weight of the particles. In further embodiments, the suspended protein particles may have a residual moisture content of 2.0 wt%, 1.0 wt%, 0.8 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, or 0.05 wt% or less, based on the total weight of the protein particles. In yet further embodiments, the residual moisture content of the suspended protein particles as defined herein may be in the range of 0.05-0.5 wt%, or 0.05-0.2 wt%, 0.1-0.5 wt%, or 0.1-0.2 wt%, based on the total weight of the particles.

[0096] Formulations in the form of suspensions containing protein particle suspensions or dispersions must be physically stable to be suitable for therapeutic use. After storage or after a period of time, the dispersed particle phase may separate from the liquid continuous phase of the suspension, for example, by particle flotation or particle sedimentation. The physical stability of a suspension may be determined, for example, by the particle settling / flotation velocity or the ease with which the particles can be redispersed.

[0097] With regard to dosing accuracy and reproducibility, particularly for injectable or parenteral suspensions, which can affect ease of injection, particles in the suspension formulation should preferably maintain a consistent particle size distribution and be easily redispersible. Furthermore, the suspension formulation should remain uniformly dispersed, and particle floating or settling should occur only at a slow rate. As used herein, the term "redispersible," which may be used interchangeably with the term "resuspensionable," etc., refers to the ability of a suspension to substantially reorganize or return to an initial or intended dispersion profile after settling or phase separation.

[0098] The suspension formulation that is insufficiently suitable and insufficiently stable tends to have poor redispersibility.Therefore, when dispersed particles undergo phase separation quickly due to particle aggregation, and particle size distribution changes over a period of time, the aggregate or cake of protein particles that may be formed cannot be easily redispersed.For example, when the size of the aggregate may cause clogging of the thin gauge needle that is usually used for subcutaneous injection, the aggregate of protein particles that is dense and poorly redispersible forms, making accurate administration difficult, and in some cases, impossible.

[0099] It has been unexpectedly found that further improvements can be achieved in the physical stability of suspension formulations comprising protein particles dispersed in a non-aqueous liquid solvent, such as a semi-fluorinated alkane, when the protein particles have a water content of at least 0.5% by weight or within the range defined above. As described herein, these suspension formulations, which can be obtained by a process comprising, for example, a spray-drying step followed by a subsequent vacuum drying step, typically have a residual water content in the range of 3-5% by weight, and it has been found that they provide suspensions that can have improved characteristics, such as redispersibility, physical stability, and injectability and / or needleability, for an even longer period of time compared to suspensions prepared from protein particles prepared solely by spray-drying.

[0100] In particular, it has been found that the dispersion properties of the suspensions, when initially prepared, are superior to suspensions prepared from the same protein particles but containing a high residual water content. It has also been found that the physical stability of the suspensions, as determined, for example, by particle size growth and redispersibility, and syringeability / passivability, can be maintained during long-term storage (e.g., up to 4, 6, or 12 months), even at elevated temperatures (stress levels) of up to 40°C.

[0101] These suspension formulations may therefore be useful as formulation and delivery vehicles for therapeutic proteins and polypeptides and / or as storage or transport vehicles for such proteins and polypeptides.

[0102] Alternatively, the water content of the suspension formulation itself may also be determined. Preferably, the total residual water content of the suspension formulation may be less than 1.0 mg / mL or less than 0.5 mg / mL based on the total volume of the formulation. In one embodiment, the total residual water content of the suspension formulation may be less than 1.0 mg / mL, less than 0.9 mg / mL, less than 0.8 mg / mL, less than 0.7 mg / mL, less than 0.6 mg / mL, less than 0.5 mg / mL, less than 0.4 mg / mL, less than 0.35 mg / mL, less than 0.3 mg / mL, less than 0.25 mg / mL, less than 0.2 mg / mL, less than 0.15 mg / mL, less than 0.1 mg / mL, or less than 0.05 mg / mL based on the total volume of the formulation. In other embodiments, the total residual water content of the suspension form may be in the range of 0.05 mg / mL to 1.0 mg / mL, or in the range of 0.005 mg / mL to 0.5 mg / mL based on the total volume of the formulation. More preferably, the total residual water content of the suspension formulation may be less than 0.1% (v / v), or less than 0.05% (v / v), based on the total volume of the formulation. In one embodiment, the total residual water content of the suspension formulation may be less than 0.1% (v / v), less than 0.09% (v / v), less than 0.08% (v / v), less than 0.07% (v / v), less than 0.06% (v / v), less than 0.05% (v / v), less than 0.04% (v / v), less than 0.03% (v / v), less than 0.02% (v / v), or less than 0.01% (v / v), based on the total volume of the formulation. In other embodiments, the total residual water content of the suspension form may be in the range of 0.001% (v / v) to 0.1% (v / v), or in the range of 0.001% (v / v) to 0.01% (v / v), based on the total volume of the formulation.

[0103] In preferred embodiments, the total solids content of the suspension is up to 30 mg / mL and the residual water content of the suspension is less than 0.15 mg / mL (less than 0.015% (v / v)) or less than 0.03 mg / mL (less than 0.003% (v / v)), based on the total volume of the formulation. In even more preferred embodiments, the total solids content of the suspension is up to 50 mg / mL and the residual water content of the suspension is less than 0.25 mg / mL (less than 0.025% (v / v)) or less than 0.05 mg / mL (less than 0.005% (v / v)), based on the total volume of the formulation. In an even more preferred embodiment, the total solids suspension is at most 100 mg / mL and the residual water content of the suspension is less than 0.5 mg / mL (less than 0.05% (v / v)) or less than 0.1 mg / mL (less than 0.01% (v / v)), based on the total volume of the formulation; in an even more preferred embodiment, the total solids suspension is at most 300 mg / mL and the residual water content of the suspension is less than 1.5 mg / mL (less than 0.15% (v / v)) or less than 0.3 mg / mL (less than 0.03% (v / v)), based on the total volume of the formulation. The residual water content of the suspension, protein particles or other components of the formulation can be determined by conventional techniques and analytical methods known in the art, such as by Karl Fischer analysis, loss on drying or thermogravimetric analysis.

[0104] The protein concentration in the suspension formulation according to the present disclosure may be 2 to 350 mg / mL, or 25 to 350 mg / mL. In other embodiments, the protein concentration may be up to 280 mg / mL, up to 210 mg / mL, up to 140 mg / mL, up to 70 mg / mL, or up to 350 mg / mL. In further embodiments, the protein concentration in the suspension formulation may be 2 to 280 mg / mL, 5 to 280 mg / mL, 25 to 280 mg / mL, 25 to 210 mg / mL, 25 to 140 mg / mL, 70 to 210 mg / mL, 70 to 280 mg / mL, 140 to 280 mg / mL, 210 to 280 mg / mL, or 5 to 50 mg / mL.

[0105] In further embodiments, the suspension formulation may have a total solids content (TSC) of 7-500 mg / mL, or 50-500 mg / mL. As understood herein, total solids content may refer to the amount of solids (mass per volume) retained after removal from the liquid phase of the formulation. In other embodiments, the total solids content of the suspension formulation may be up to 500 mg / mL. In further embodiments, the total solids content of the suspension formulation may be up to 400 mg / mL, up to 350 mg / mL, up to 300 mg / mL, up to 200 mg / mL, or up to 100 mg / L. In yet further embodiments, the total solids content may be 7-450 mg / mL, 25-450 mg / mL, 50-400 mg / mL, 50-300 mg / mL, 100-300 mg / mL, 100-200 mg / mL, or 200-300 mg / mL.

[0106] In one embodiment, a suspension formulation according to any one of the other embodiments or combinations of embodiments described herein may contain 50-70% protein based on the total solids content (TSC) of the formulation. In other embodiments, the amount of protein based on the total solids content of the suspension formulation may be about 50%, about 55%, about 60%, about 65%, or about 70%. In further embodiments, the amount of protein based on the total solids content of the suspension formulation may be 50-60%, 55-65%, or 60-70%.

[0107] Protein particles according to the present disclosure preferably have an average particle size of less than 30 μm or less than 50 μm when determined by laser diffraction. In further embodiments, the average particle size of the protein particles may be less than 20 μm, less than 15 μm, less than 10 μm, or less than 5 μm when determined using laser diffraction. Optionally, the basis of particle size distribution by which the particle size of a suspension formulation may be determined using laser diffraction may be volume; in other words, "average particle size" may refer to the volume average diameter. In any embodiment, the protein particles may have a volume average diameter of less than 50 μm, less than 30 μm, less than 20 μm, less than 15 μm, less than 10 μm, or less than 5 μm.

[0108] In further embodiments, a suspension formulation according to the present disclosure can be comprised of protein particles, wherein at least 90% of the protein particles have an average particle size of 1-30 μm, or 1-50 μm, as determined by laser diffraction. Optionally, the basis of particle size distribution for determining the particle size of the suspension formulation by laser diffraction can be volume, i.e., "average particle size" can refer to the volume average diameter. Optionally, at least 90% of the protein particles in the suspension formulation can have a volume average diameter of less than 50 μm, less than 30 μm, less than 20 μm, less than 15 μm, less than 10 μm, or less than 5 μm.

[0109] In one embodiment, a suspension formulation according to the present disclosure may consist of protein particles consisting essentially of a protein, a stabilizer, and optionally one or more excipients. In a further embodiment, the suspension formulation consists essentially of protein particles suspended in a non-aqueous solvent, the protein particles consisting of a protein and a stabilizer, and optionally one or more excipients.

[0110] In one embodiment, the suspension formulation may further comprise one or more excipients as defined above, such as a surfactant, e.g., polysorbate 80 or polysorbate 20. In another embodiment, the suspension formulation according to the present disclosure does not comprise a surfactant and / or a preservative, or does not comprise any surfactant and / or preservative. A preservative may be any excipient added as an antimicrobial agent to prevent bacterial contamination and growth in the formulation. Examples of preservatives include benzalkonium chloride, 1,3-butanediol, phenol, and benzyl alcohol.

[0111] In a further embodiment, the present disclosure relates to a suspension formulation comprising protein particles suspended in a non-aqueous solvent comprising, or consisting essentially of, a semi-fluorinated alkane, wherein the protein particles have a relative weight ratio of protein to stabilizer of 1:1 to 7:3, the residual water content of the protein particles is less than 0.5 wt%, preferably less than 0.3 wt%, based on the total weight of the protein particles, and the total solids content of the formulation is about 300 mg / mL or less.

[0112] In such embodiments, the semi-fluorinated alkane is preferably selected from F4H5 or F6H8. In one embodiment, the total solids content of the formulation is 300 mg / mL. In an alternative embodiment, the total solids content of the formulation is about 100 mg / mL or less. In a further aspect of this embodiment, the residual water content of the formulation may be less than 0.4 wt.%, or less than 0.25 wt.%, based on the total weight of the formulation. The protein particles according to any one of these embodiments are preferably spray-dried protein particles, and more preferably spray-dried and vacuum-dried protein particles.

[0113] The suspension formulation, in another embodiment, is comprised of protein particles suspended in a non-aqueous solvent, the non-aqueous solvent consisting essentially of a semi-fluorinated alkane selected from F4H5 or F6H8 (or a mixture thereof), and optionally one or more excipients, preferably the one or more excipients are solubilized or soluble in F4H5 or F6H8, the protein particles being spray-dried (and vacuum-dried) particles comprising the protein, a stabilizer, and optionally one or more further excipients (e.g., a buffer such as histidine), and the protein is the protein is a monoclonal antibody or is selected from the group consisting of lysozyme, immunoglobulin, aflibercept, ziv-aflibercept, or bevacizumab; the stabilizer is a sugar, preferably selected from sucrose and trehalose, and / or a polyol; the relative weight ratio of protein to stabilizer in the protein particles is 1:1 to 7:3; the total solids content of the formulation is about 300 mg / mL or less; and the residual water content of the protein particles is 0.5 wt.%, preferably less than 0.3 wt.%, based on the total weight of the protein particles.

[0114] Suspension formulations according to the present disclosure may be administered by injection or parenteral administration. In one embodiment, the suspension may be drawn (aspirated) into a syringe and injected through a fine gauge needle (e.g., a 27G or 23G needle). In one embodiment, the suspension formulation may be injected with an injection gliding force of less than 35 Newtons (N). In a further embodiment, the formulation may be injected with a gliding force of less than 15 Newtons (N). In yet further embodiments, the injection gliding force of the formulation may be less than 25 Newtons, less than 20 Newtons, less than 15 Newtons, or less than 10 Newtons, or between 1 and 10 Newtons, or between 5 and 15 Newtons. Preferably, the injection gliding force of the suspension formulation remains substantially unchanged over a storage period of at least up to 12 months. In further embodiments, the injection gliding force required to administer the suspension formulation may be less than 35 N, less than 25 N, less than 20 N, less than 15 N, or less than 10 N, between 1 and 35 N, or between 5 and 35 N after storage of the suspension formulation at 40° C. for at least up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0115] In a specific embodiment, the suspension formulation comprises a non-aqueous solvent comprising or consisting essentially of a semi-fluorinated alkane, preferably F4H5 or F6H8, and has a pouring glide force of less than 15 N, or less than 10 N, or between 1 and 15 N, or between 5 and 15 N. Preferably, the pouring glide force of the suspension formulation remains substantially unchanged over a storage period of at least up to 12 months. In further embodiments, the formulation may have a pouring glide force of at least less than 15 N, or less than 14 N, less than 13 N, less than 12 N, less than 11, or less than 10 N, or between 1 and 15 N after storage of the suspension formulation at 40° C. for up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0116] In another embodiment, the suspension formulation comprises a non-aqueous solvent comprising or consisting essentially of ethyl oleate or ethyl lactate and has a pouring gliding force of less than 20 N. Preferably, the pouring gliding force of the suspension formulation remains substantially unchanged over a storage period of at least up to 12 months. In further embodiments, the formulation may have a pouring gliding force of less than 20 N, or between 5 and 20 N, after storage of the suspension formulation at 40° C. for at least up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0117] As understood herein, injection glide force refers to the maximum force required to inject the formulation through a needle and syringe. In any of these above embodiments, the injection glide force may be applicable to achieve an injection with a flow rate of 0.1 mL / sec and a 1 mL syringe and a 27 G needle (approximately 210 μm internal diameter) of a type corresponding to or similar to the syringes and needles exemplified herein.

[0118] Suspension formulations according to the present disclosure preferably have a viscosity of 5 to 40 mPa·s when measured by a rotational viscometer at 25° C. In some embodiments, suspension formulations according to the present disclosure may have a viscosity of 10 to 30 mPa·s, 10 to 25 mPa·s, 10 to 20 mPa·s, 15 to 30 mPa·s, 15 to 25 mPa·s, or 15 to 30 mPa·s when measured by a rotational viscometer at 25° C. In other embodiments, suspension formulations may have a viscosity of about 10 mPa·s, about 15 mPa·s, about 20 mPa·s, about 25 mPa·s, about 30 mPa·s, or less than 40 mPa·s, less than 35 mPa·s, less than 30 mPa·s, less than 25 mPa·s, less than 20 mPa·s, less than 15 mPa·s, or less than 10 mPa·s when measured by a rotational viscometer at 25° C.

[0119] Upon settling or suspending, particles of a suspension formulation according to the present disclosure may be resuspended, such as by rotation (e.g., using a vertical rotor), shaking by hand, or shaking at a frequency of up to 15 Hz. In one embodiment, a suspension formulation described herein may be redispersed by shaking at a frequency of up to 5 Hz, or 10 Hz, or 2-15 Hz, 2-10 Hz, 2-5 Hz, 5-15 Hz, or 5-10 Hz. Shaking may be performed using devices known in the art, such as those described herein, to redisperse suspensions.

[0120] In one embodiment, the suspension formulation may be redispersible after storage at room temperature for at least one month. In further embodiments, the suspension formulation may be redispersible for at least one month, three months, six months, or twelve months. As used herein, the term "redispersible," which may be used interchangeably with the term "resuspensionable," refers to the ability of a suspension to substantially reorganize or return to its initial or intended dispersion profile after settling or phase separation, for example, during storage. In further embodiments, formulations according to the present disclosure may be redispersible after storage at up to 40° C. for at least one month, three months, six months, or twelve months.

[0121] In another embodiment, the suspension formulation may be redispersed or resuspended in less than 1000 seconds and retain at least 70%, 80%, or 90% of the original particle size distribution. Preferably, the suspension formulation may be redispersed or resuspended in less than 1000 seconds and retain at least 70%, 80%, or 90% of the original d90 particle size distribution. The time period refers to the time required to redisperse a settled or phase-separated suspension, such as by mechanical or physical means, such as those described herein (e.g., shaking by hand or using a shaker). In other embodiments, resuspension of a suspension formulation according to the present disclosure may occur in less than 800 seconds, or less than 900 seconds, or within a period of 2 to 800 seconds, or within a period of 2 to 900 seconds, or within a period of 2 to 1000 seconds. In yet further embodiments, the suspension formulation comprises a non-aqueous solvent, the non-aqueous solvent being a semi-fluorinated alkane, and the suspension formulation redisperses in less than 50 seconds and retains at least 70%, 80%, or at least 90% of its original particle size distribution. In further embodiments, the suspension formulation may be redispersible in less than 20 seconds, or less than 30 seconds, or within 2-50 seconds, or within 2-30 seconds, or within 2-20 seconds. In yet other embodiments, the formulation may be redispersible within 30 seconds at a frequency of 5 Hz or within 30 seconds by manual shaking to retain at least 70%, 80%, or 90% of its original particle size distribution. In yet further embodiments, the suspension formulation comprises a non-aqueous solvent, the non-aqueous solvent being a medium-chain triglyceride (MCT), ethyl oleate, or ethyl lactate, and the formulation may be redispersible in less than 1000 seconds to retain at least 70%, 80%, or 90% of its original particle size distribution. In a more specific embodiment, the non-aqueous solvent may be MCT, and the suspension formulation is redispersible in less than 800 seconds, or less than 900 seconds, or within 200 to 1000 seconds, 200 to 900 seconds, 200 to 800 seconds, 300 to 1000 seconds, 300 to 900 seconds, or 300 to 800 seconds.

[0122] In one embodiment, the protein particles are spray-dried or freeze-dried protein particles. As used herein, the term "spray-dried" refers to protein particles prepared using a spray-drying process, which includes spray-drying an aqueous solution containing a protein and a stabilizer, and optionally one or more additional excipients. The term "lyophilized" refers to protein particles prepared by freeze-drying an aqueous solution containing a protein and a stabilizer, and optionally one or more additional excipients. In a preferred embodiment, the protein particles suspended in a non-aqueous solvent described herein are spray-dried protein particles. In a further embodiment, the protein particles suspended in a non-aqueous solvent described herein are spray-dried and additionally dried protein particles, i.e., protein particles obtained by spray-drying and then subjected to an additional drying step, such as an additional vacuum drying step. In yet another embodiment, the protein particles may be freeze-dried and additionally dried protein particles, i.e., protein particles obtained by freeze-drying and then subjected to an additional drying step, such as an additional vacuum drying step. As understood herein, the term vacuum-dried refers to protein particles that have been subjected to a vacuum drying process. Vacuum drying can be distinguished from freeze drying in that the particles are not dried under cryogenic conditions as practiced in the art for freeze drying.

[0123] Because the spray-drying process requires exposing the protein particles to high temperatures and may therefore be associated with loss of protein or protein activity, freeze-drying (lyophilization) is a typical and often preferred method for removing water from protein particles. However, protein particles according to the present disclosure have been prepared under conditions that do not sublimate water, such as lyophilization, by using a spray-drying process in combination with a vacuum drying process carried out at about ambient temperature or higher (e.g., in the range of 15-40°C), and have been observed to not only provide a physically stable suspension in a non-aqueous solvent of protein particles with a uniform particle size distribution suitable for administration by injection, but also to retain protein activity (see Example 2).

[0124] In a further aspect, the present disclosure relates to the use of the suspension formulations described herein for therapeutic and / or diagnostic applications. The suspension formulations can be used, for example, to treat a disease or condition affecting the skin, eyes, ears, nose, or lungs in a subject in need thereof. As understood herein, "subject" may refer to a human subject and may be used synonymously with the term "patient." The subject or patient may be suffering from or diagnosed with a disease or condition and may be in need of treatment or relief, amelioration, control, or regulation of the disease or condition, or the progression of one or more symptoms of the condition or disease, or prevention of the occurrence of the disease or condition. Optionally, the subject may also be a veterinary subject.

[0125] In one embodiment, the suspension formulation may be used to treat an ocular disease or condition, e.g., affecting one or both eyes of a subject. The suspension formulations described herein may be administered topically (e.g., to a tissue or organ surface) or by injection. The formulation may be administered parenterally, e.g., by injection, e.g., subcutaneous or intramuscular. In one embodiment, the formulation may be administered by injection into the eye (intraocular injection) or into an ocular tissue. Methods of intraocular injection applicable in the context of the present disclosure include intravitreal, suprachoroidal, juxtascleral, subconjunctival, intracameral, subretinal, subtenon, or periocular injection.

[0126] Use of the suspension formulation described in any one of the embodiments herein is also provided in the context of the present disclosure for the manufacture or preparation of a drug or medicament for such use. Similarly, the therapeutic use described in any one of the embodiments described herein above or a combination thereof may be featured in a method of treating a subject in need thereof, comprising administering to the subject the suspension formulation.

[0127] In a still further aspect, the present disclosure relates to a kit comprising a suspension formulation as defined herein and a container adapted to hold said formulation, and optionally dispensing means.

[0128] An example of a dispensing means may be a dispensing means adapted to administer a suspension formulation topically, or a dispensing means adapted to administer by injection, for example, to the skin, eye, ear, nose, or lungs of a subject. Examples of dispensing means include, for example, a needle suitable for or adapted to inject the formulation, or an eyedropper adapted to dispense a suspension formulation into the eye. In one embodiment, a container adapted to hold a suspension formulation may also be adapted to resuspend the protein particles. The container may be suitable for or adapted for mechanical agitation, for example, by rotating or shaking by hand (manual), or for agitation means such as a stirrer or rotor. A container according to any one of the embodiments herein may also be adapted for shaking at a frequency of up to 15 Hz. The containers described herein may also be adapted and filled to provide sufficient headspace to allow resuspension of the formulation. The container may also be adapted, in some embodiments, to administer the formulation by injection or topical administration. In one embodiment, the container and optionally the dispensing means are adapted for topical administration or parenteral injection.

[0129] In one embodiment, the kit may be a pre-filled syringe, which may function as a container adapted to hold the formulation, and optionally includes a needle for injection.In yet another embodiment, the syringe and dispensing means may be adapted for intraocular injection, preferably for intravitreal injection, suprachoroidal injection, juxtascleral injection, subconjunctival injection, intracameral injection, subretinal injection, subtenon injection, or periocular injection.The kit may also further include instructions for the container or dispensing means and instructions for administering the suspension formulation, which may be provided in a tangible or readable form, such as an instruction leaflet, or a label or insert in the package.

[0130] The following list of numbered items are embodiments encompassed by the present invention:

[0131] 1. A suspension formulation comprising protein particles suspended in a non-aqueous solvent, the particles comprising a protein and a stabilizer, wherein the residual water content of the suspended protein particles is less than 1.0% by weight, based on the total weight of the particles.

[0132] 2. The suspension formulation according to item 1, comprising spray-dried or freeze-dried protein particles.

[0133] 3. The suspension formulation according to item 2, comprising spray-dried and vacuum-dried protein particles.

[0134] 4. The suspension formulation according to any one of items 1 to 3, wherein the non-aqueous solvent is liquid at room temperature and / or immiscible with water.

[0135] 5. The suspension formulation according to any one of items 1 to 4, wherein the non-aqueous solvent comprises a semi-fluorinated alkane, a medium-chain triglyceride (MCT), ethyl lactate, ethyl oleate, or a mixture thereof.

[0136] 6. The suspension formulation according to any one of items 1 to 5, wherein the non-aqueous solvent comprises one or more semi-fluorinated alkanes.

[0137] 7. The suspension formulation according to any one of items 1 to 6, wherein the non-aqueous solvent consists of one or more semi-fluorinated alkanes and, optionally, one or more pharmaceutically acceptable excipients.

[0138] 8. One or more semifluorinated alkanes have the formula F(CF2) n (CH2) m wherein n is an integer selected from 4 to 6, and m is an integer selected from 4 to 8.

[0139] 9. The suspension formulation according to item 8, wherein the non-aqueous solvent comprises or consists of one or more semi-fluorinated alkanes selected from the group consisting of F4H4, F4H5, F4H6, F4H8, F6H4, F6H6 and F6H8.

[0140] 10. The suspension formulation according to any one of items 1 to 9, wherein the non-aqueous solvent is a solvent selected from F4H5, F6H8, ethyl oleate and medium-chain triglycerides, or a solvent selected from F4H5 and F6H8.

[0141] 11. The suspension formulation according to any one of items 1 to 10, wherein the residual water content of the suspended protein particles is less than 0.5% by weight, based on the total weight of the particles.

[0142] 12. The suspension formulation according to any one of items 1 to 11, wherein the residual water content of the suspended protein particles is in the range of 0.05 to 1.0 wt. %, based on the total weight of the particles.

[0143] 13. The suspension formulation according to any one of items 1 to 12, wherein the residual water content of the suspended protein particles is in the range of 0.05 to 0.5% by weight, based on the total weight of the particles.

[0144] 14. The suspension formulation according to any one of items 1 to 13, wherein the residual water content of the suspended protein particles is less than 1.0 mg / mL, or less than 0.5 mg / mL, based on the total volume of the formulation.

[0145] 15. The suspension formulation according to any one of items 1 to 14, wherein the relative weight ratio of protein to stabilizer is in the range of 1:1 to 7:3.

[0146] 16. The suspension formulation according to any one of items 1 to 15, wherein the stabilizer is selected from sugars, polyols, amino acids, amines, surfactants, antioxidants, polymers, salts or combinations thereof.

[0147] 17. The suspension formulation according to item 16, wherein the stabilizer is selected from sugars, polyols, amino acids, amines, glycols and inorganic salts.

[0148] 18. The suspension formulation according to any one of items 1 to 17, wherein the stabilizer is a sugar, a polyol, a polysorbate, or a combination thereof.

[0149] 19. The suspension formulation according to any one of items 1 to 18, wherein the stabilizing agent is a sugar, preferably a sugar selected from trehalose and sucrose.

[0150] 20. The suspension formulation according to any one of items 1 to 19, wherein the protein has a molecular mass of 10 to 300 kDa.

[0151] 21. The suspension formulation according to any one of items 1 to 20, wherein the protein is selected from an antigen-binding polypeptide or protein, a vaccine and an enzyme.

[0152] 22. The suspension formulation according to any one of items 1 to 21, wherein the protein is selected from an antibody, preferably a monoclonal antibody or an immunoglobulin (IgG), an antibody fragment, a fusion protein comprising an antibody fragment, an antibody-drug conjugate, and an enzyme.

[0153] 23. The suspension formulation according to any one of items 1 to 22, wherein the protein is a chimeric monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody.

[0154] 24. The suspension formulation according to any one of items 1 to 23, wherein the protein is selected from the group consisting of lysozyme and an antibody (e.g., aflibercept, ziv-aflibercept, or bevacizumab).

[0155] 25. The suspension formulation according to any one of items 1 to 24, wherein the protein is selected from the group consisting of aflibercept, ziv-aflibercept, and bevacizumab.

[0156] 26. The suspension formulation according to any one of items 1 to 25, having a protein concentration of 5 to 350 mg / mL.

[0157] 27. The suspension formulation according to any one of items 1 to 26, wherein the total solid content (TSC) of the formulation is 10 to 500 mg / mL.

[0158] 28. The suspension formulation according to any one of items 1 to 27, wherein the ratio of protein to total solid content (TSC) of the formulation is 50 to 70%.

[0159] 29. The suspension formulation according to any one of items 1 to 28, wherein the protein particles have an average particle size of less than 30 μm as determined by laser diffraction.

[0160] 30. The suspension formulation according to any one of items 1 to 29, wherein at least 90% of the protein particles have an average particle size of 1 to 30 μm as determined by laser diffraction.

[0161] 31. The suspension formulation according to any one of items 1 to 30, having an injection gliding force of less than 35 N, preferably less than 25 N after storage at 40°C for up to 12 months.

[0162] 32. The suspension formulation according to item 31, wherein the non-aqueous solvent comprises or consists of a semi-fluorinated alkane, preferably F4H5, or F6H8, and has a pouring gliding force of less than 15 N, preferably less than 15 N after storage at 40°C for up to 12 months.

[0163] 33. The suspension formulation according to any one of items 31 or 32, wherein the injection gliding force is for injection with a flow rate of 0.1 mL / sec and a 1 mL syringe and a 27 G needle.

[0164] 34. The suspension formulation according to any one of items 1 to 33, wherein the viscosity of the formulation is 5 to 40 mPa s when measured with a rotational viscometer at 25°C.

[0165] 35. The suspension formulation according to any one of items 1 to 34, wherein the protein particles consist of a protein, a stabilizer, and optionally one or more excipients.

[0166] 36. The suspension formulation according to any one of items 1 to 35, wherein the suspension formulation consists of protein particles suspended in a non-aqueous solvent, the protein particles consisting of a protein and a stabilizer, and optionally one or more excipients.

[0167] 37. The suspension formulation according to any one of items 1 to 36, wherein the formulation further comprises one or more excipients, such as, for example, a surfactant (e.g., polysorbate 20, or polysorbate 80).

[0168] 38. The suspension formulation according to any one of items 1 to 37, wherein the suspension formulation does not contain surfactants and / or preservatives or does not contain any surfactants and / or preservatives.

[0169] 39. the non-aqueous solvent comprises or consists of a semi-fluorinated alkane, preferably selected from F4H5 or F6H8, - the relative weight ratio of protein to stabilizer in the protein particles is 1:1 to 7:3; the residual water content of the protein particles is less than 0.5% by weight, preferably less than 0.3% by weight, relative to the total weight of the protein particles; 39. The suspension formulation according to any one of items 1 to 38, wherein the total solids content of the formulation is not more than about 300 mg / mL.

[0170] 40. The suspension formulation according to item 39, wherein the total solids content of the formulation is 300 mg / mL.

[0171] 41. The suspension formulation according to item 39, wherein the total solids content of the formulation is about 100 mg / mL or less.

[0172] 42. The suspension formulation according to any one of items 39 to 41, wherein the residual water content of the formulation is less than 0.4% by weight, preferably less than 0.25% by weight, based on the total weight of the formulation.

[0173] 43. The suspension formulation according to any one of items 39 to 42, wherein the protein particles are spray-dried protein particles, preferably spray-dried and vacuum-dried protein particles.

[0174] 44.- The non-aqueous solvent consists of a semi-fluorinated alkane selected from F4H5 or F6H8, and optionally one or more excipients; the protein particles are spray-dried particles comprising a protein, a stabilizer, and optionally one or more further excipients, wherein the protein is a monoclonal antibody or the protein is selected from the group consisting of lysozyme, immunoglobulin, aflibercept, ziv-aflibercept, or bevacizumab, and the stabilizer is a sugar, preferably selected from sucrose and trehalose; - the relative weight ratio of protein to stabilizer in the protein particles is 1:1 to 7:3; the residual water content of the protein particles is less than 0.5% by weight, preferably less than 0.3% by weight, relative to the total weight of the protein particles; 44. The suspension formulation according to any one of items 39 to 43, wherein the total solids content of the formulation is not more than about 300 mg / mL.

[0175] 45. The suspension formulation according to any one of items 1 to 44 for use as a medicament.

[0176] 46. ​​A suspension formulation for use according to item 45, wherein the use includes treatment of a disease or condition affecting the skin, eyes, ears, nose, or lungs in a subject in need thereof.

[0177] 47. A suspension formulation for use according to item 46, wherein the use comprises the treatment of an eye disease or condition.

[0178] 48. A suspension formulation for use according to items 45 to 47, wherein the formulation is administered topically or by injection.

[0179] 49. A suspension formulation for use according to any one of items 45 to 48, wherein the formulation is administered by injection (e.g. subcutaneous or intramuscular injection) or by injection into the eye (intraocular injection), preferably by intravitreal, suprachoroidal, juxtascleral, subconjunctival, intracameral, subretinal, subtenon, or periocular injection.

[0180] 50. Use of a suspension formulation according to any one of items 1 to 44 in the manufacture of a medicament for treating a disease or condition in a subject or patient.

[0181] 51. The use according to item 50, wherein the medicament is for use in the treatment of a disease or condition affecting the skin, eyes, ears, nose, or lungs in a subject.

[0182] 52. The use according to item 51, wherein the drug is for use in treating an eye disease or condition.

[0183] 53. Use according to any one of items 50 to 52, wherein the drug is a drug to be administered locally or a drug formulated or adapted for injection.

[0184] 54. The use according to any one of items 50 to 53, wherein the drug is administered by injection (e.g. subcutaneous or intramuscular injection) or by injection into the eye (intraocular injection), preferably by intravitreal, suprachoroidal, juxtascleral, subconjunctival, intracameral, subretinal, subtenon, or periocular injection.

[0185] 55. A method for treating a disease or condition, comprising administering to a subject in need thereof a suspension formulation according to any one of items 1 to 44.

[0186] 56. The method of item 55, wherein the disease or condition is a disease or condition that adversely affects the skin, eyes, ears, nose, or lungs of the subject.

[0187] 57. The method according to item 55 or 56, wherein the disease or condition is an eye disease or condition.

[0188] 58. The method according to any one of items 55 to 57, wherein the suspension formulation is administered topically or by injection.

[0189] 59. The method according to any one of items 55 to 58, wherein the suspension formulation is administered by injection (e.g. subcutaneous or intramuscular injection) or by injection into the eye (intraocular injection), preferably by intravitreal, suprachoroidal, juxtascleral, subconjunctival, intracameral, subretinal, subtenon, or periocular injection.

[0190] 60. A suspension formulation as defined in any one of items 1 to 44 obtained or obtainable by a process, which process comprises: a) spray-drying or freeze-drying an aqueous solution containing a protein and a stabilizer to obtain protein particles; b) drying the protein particles obtained in step a) to obtain a residual moisture content of less than 1.0 wt. % or less than 0.5 wt. %, based on the total weight of the particles; and c) suspending the protein particles of step b) in a non-aqueous solvent; d) and optionally homogenizing the suspension, preferably by high shear homogenization, milling or sonication.

[0191] 61. A suspension formulation as defined in any one of items 1 to 44 obtained or obtainable by a process, which process comprises: a) spray-drying or freeze-drying an aqueous solution containing a protein and a stabilizer to obtain protein particles; b) vacuum drying the protein particles obtained in step a); and c) suspending the protein particles of step b) in a non-aqueous solvent; d) and optionally homogenizing the suspension, preferably by high shear homogenization, milling or sonication.

[0192] 62. A suspension formulation obtainable by the process according to item 60 or 61, comprising in step a) spray-drying an aqueous solution comprising the protein and the stabilizer to obtain protein particles.

[0193] 63. A suspension formulation obtainable according to any one of items 60 or 62, wherein the relative weight ratio of protein to stabilizer is from 1:1 to 7:3.

[0194] 64. A suspension formulation obtainable by the process according to any one of items 60 to 63, wherein the spray drying of step a) is carried out using a cyclone spray dryer.

[0195] 65. The suspension formulation obtainable according to any one of items 60 to 64, wherein the drying step of step b) is a vacuum drying step, preferably the vacuum drying step is carried out at a temperature of 15 to 40°C and at a pressure of 0.01 to 100 mbar.

[0196] 66. A suspension formulation obtainable according to any one of items 60 to 65, wherein step b) is carried out for at least 12 hours, or at least 24 hours.

[0197] 67. The suspension formulation obtainable according to any one of items 60 to 66, wherein the vacuum drying step of step b) is carried out to obtain particles having a residual moisture content of less than 1.0% by weight, or less than 0.5% by weight, based on the weight of the particles.

[0198] 68. A process for producing a suspension formulation as defined in any one of items 1 to 44, the process comprising: a) spray-drying or freeze-drying an aqueous solution containing a protein and a stabilizer to obtain protein particles; b) drying the protein particles obtained in step a) to obtain particles with a residual moisture content of less than 1.0 wt. % or less than 0.5 wt. %, based on the total weight of the particles; and c) suspending the protein particles of step b) in a non-aqueous solvent, and optionally d) homogenizing the suspension formulation, preferably by high shear homogenization, milling or sonication.

[0199] 69. A process for producing a suspension formulation as defined in any one of items 1 to 44, the process comprising: a) spray-drying or freeze-drying an aqueous solution containing a protein and a stabilizer to obtain protein particles; b) vacuum drying the protein particles obtained in step a); and c) suspending the protein particles of step b) in a non-aqueous solvent, and optionally d) homogenizing the suspension formulation, preferably by high shear homogenization, milling or sonication.

[0200] 70. The process according to any one of items 68 or 69, comprising (in step a) spray-drying an aqueous solution comprising the protein and the stabilizer to obtain protein particles.

[0201] 71. The process according to any one of items 68 to 70, wherein the relative weight ratio of protein to stabilizing agent is 1:1 to 7:3.

[0202] 72. The process according to any one of items 68 to 71, wherein the drying step of step b) is a vacuum drying step, preferably the vacuum drying step is carried out at a temperature of 15 to 40°C and a pressure of 0.01 to 100 mbar.

[0203] 73. The process according to any one of items 68 to 72, wherein step b) is carried out for at least 12 hours, or at least 24 hours.

[0204] 74. The process according to any one of items 68 to 73, wherein the vacuum drying step of step b) is carried out to obtain particles having a residual moisture content of less than 1.0 wt. %, or less than 0.5 wt. %, based on the weight of the particles.

[0205] 75. A kit comprising a suspension formulation as defined in any one of items 1 to 44, and a container adapted to hold the formulation, and optionally dispensing means.

[0206] 76. The kit according to item 75, wherein the container adapted to hold the formulation is a prefilled syringe, and the kit further comprises the syringe and optionally a dispensing means, preferably a needle adapted for injecting the formulation.

[0207] 77. The kit according to item 76, wherein the syringe and dispensing means are adapted for intraocular injection, preferably for intravitreal, suprachoroidal, juxtascleral, subconjunctival, intracameral, subretinal, subtenon, or periocular injection.

[0208] 78. A dosing device containing a suspension formulation as defined in any one of items 1 to 44.

[0209] 79. The administration device according to item 78, wherein the administration device is adapted to administer the suspension formulation by topical administration or injection.

[0210] 80. An administration device according to item 78 or 79, wherein the administration device comprises a syringe and, optionally, a needle.

[0211] 81. The administration device according to any one of items 78 to 80, wherein the administration device is adapted for subcutaneous administration of a suspension formulation as defined in any one of items 1 to 44.

[0212] 82. The process according to items 68 to 74, further comprising the step of selecting protein particles having a predetermined particle size and suspended in a non-aqueous solvent.

[0213] 83. The process according to item 82, wherein the predetermined particle size is characterized by a distribution of at least 90% of particles having an average particle size of 1 to 15 μm, 1 to 30 μm, or 1 to 50 μm, or characterized by an average particle size of less than 50 μm, less than 30 μm, less than 15 μm, 1 to 15 μm, 1 to 30 μm, or 1 to 50 μm, respectively, as determined by laser diffraction.

[0214] The following examples serve to illustrate the present invention but should not be construed as limiting the scope of the invention. [Example]

[0215] Example 1 Preparation of suspension formulation Materials. A bulk solution of lysozyme was prepared by dissolving pure lysozyme (lys) (Ovobest, Neuenkirchen-Voerden, Germany) in 10 mM histidine buffer at pH 6.0. A model monoclonal antibody (mAb) of IgG1 thyme was used, at 56 mg / mL in 25 mM histidine, 1.6 mM glycine buffer at pH 6.0. The mAb was produced in CHO cells, which yielded 1.49 mL g -1 ·cm -1The ε at 280 nm was 0.01. A sample of bevacizumab (Beva) (commercially available as Avastin) was obtained from a local pharmacy. Trehalose (Tre) (Hayashibara Co. Ltd., Okayama, Japan), sucrose (Suc), L-histidine, L-histidine hydrochloride monohydrate (Sigma-Aldrich, St. Louis, USA), and polysorbate 20 (PS20) (Merck KGaA, Darmstadt, Germany) were used to prepare the formulation in highly purified water prepared with an ELGA Purelab system (ELGA LabWater, Celle, Germany). Perfluorobutylpentane (F4H5) and perfluorohexyloctane (F6H8) were provided by Novaliq GmbH (Heidelberg, Germany). Additionally, medium chain triglycerides (MCT) (Miglyol 812 from Caesar & Loretz GmbH, Hilden, Germany) and ethyl oleate (EO) (Sigma-Aldrich, St. Louis, USA) were also tested as suspension solvents.

[0216] Analysis method UV-Vis-protein concentrations were measured at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA).

[0217] The powders were examined by scanning electron microscopy (SEM) using an FEI Helios G3 UC (Thermo Fisher Scientific, Waltham, USA) on self-adhesive carbon tape placed on an aluminum stub. The suspended and milled powder (F6H8) was pipetted directly onto the carbon tape and dried in a VTS-2 vacuum oven (Memmert, Schwabach, Germany) at 10 mbar for 24 h.

[0218] Laser diffraction - Particle size distribution was analyzed using a Horiba laser diffraction particle size analyzer LA-960 (Horyiba, Kyoto, Japan) in isooctane containing 1% Span 80 as the dispersant. To examine the initial dispersibility, no additional dispersion step was performed in the dispersant. After an additional dispersion step using a Bandelin Sonoplus ultrasonic homogenizer (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) equipped with an MS72 probe (20% intensity for 30 seconds), particle size was analyzed after storage. An additional step was performed to distinguish between agglomerated particles and particles that had already been sintered together.

[0219] Optical microscopy was performed at 200x magnification using a Keyence Digital microscope VHX500F (Keyence Corporation, Osaka, Japan) equipped with a VH-Z100R lens. For analysis, the suspension was dispersed in MCT to a concentration of 5 mg / mL. The resulting suspension was transferred onto a glass slide and then examined.

[0220] Preparation of suspension formulation Spray drying - Feed solutions for spray drying were prepared with a total solids content of 7.5% (m / V) and containing trehalose or sucrose at different protein-to-stabilizer ratios, and optionally a surfactant (e.g., polysorbate 20). Protein particles based on lysozyme (lys), a model monoclonal antibody (mAb), and bevacizumab (beva) were prepared. The protein-to-stabilizer ratios listed are based on mass ratios. All solutions were prepared in 10 mM histidine buffer at pH 6.0.

[0221] Follow the manufacturer's recommended standards (e.g., nozzle diameter 0.7 mm, dry air flow rate 35 m 3 Spray drying was carried out using a Büchi B290 (Büchi AG, Flawil, Switzerland) equipped with a high-performance cyclone according to the following conditions: suction pressure 414 L / h, atomizing air flow rate 414 L / h, while the outlet temperature was kept at 70°C.

[0222] The protein-containing particles obtained from spray drying were transferred to a single 10R glass vial (MGlas AG, Münnerstadt, Germany) and fitted with a lyophilization stopper (Helvoet Pharma, Tilburg, Netherlands). An additional drying step involved vacuum drying at 32 °C and 0.1 mbar for 24 h using a Christ 2-6D (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany).

[0223] To prevent the hygroscopic powder from taking up water, the processing of the protein-containing particles was carried out under a nitrogen atmosphere.

[0224] Suspension formulations were prepared using spray-dried and vacuum-dried protein-containing formulations. Suspension formulations were prepared at different concentrations in a single glass vial of type 2R (Schott AG, Mainz, Germany) (Beva), type 6R (mAb), or type 20R (Lys) (MGlas AG, Münnerstadt, Germany) by adding each solvent to a weighed amount of spray-dried protein-containing particles. The suspension was then homogenized using either a high-shear homogenizer, Ultraturrax T10 (IKA-Werke GmbH & Co. KG, Staufen, Germany) (sh; 2 min / 20,000 rpm) or an ice-cooled VWR Ultrasonic cleaner (VWR, Radnor, USA) (us; 5 min, 10 min, and 15 min, followed by an additional 20 min with manual shaking).

[0225] Suspension concentrations are based on total suspension solids (TSC).

[0226] The amount of residual water in protein-containing particles or suspension formulations was analyzed using a Karl-Fischer-Titrator Aqua 40.00 equipped with a headspace module (Analytik Jena AG; Jena, Germany) at a chamber temperature of 100°C.

[0227] Following these general methods, exemplary suspension formulations 1a-15b are listed in Table 1. Table 1 lists the protein to stabilizer ratio for each formulation, as well as the determined residual water content of the particles and suspension formulation.

[0228] JPEG0007761562000001.jpg223170

[0229] Redispersion of particles after suspension preparation For the preparation of injectable suspension formulations, it is very important to obtain a uniform suspension formulation at the initial stage.The suspension formulation can be homogenized using suitable dispersion techniques, such as high-shear homogenizer, suspension grinding, or ultrasonic treatment techniques.Herein, the suspension formulation was prepared using an ice-cooled ultrasonic bath, or alternatively, using a high-shear homogenizer.

[0230] The quality of the suspension in terms of dispersibility was observed to be higher for suspension formulations prepared by an additional drying step after spray drying.

[0231] Suspension formulations prepared with additionally dried protein-containing particles using ultrasonic bath homogenization were found to be readily dispersible for F4H5, F6H8, EO, and MCT (formulations 1a-d). This was also observed for the high-concentration formulation (2; 300 mg / mL) dispersed in F6H8 as the liquid vehicle, the formulation containing PS20 (polysorbate 20) (3), and the formulation with a high trehalose content (4).

[0232] It was observed directly after preparation that suspensions prepared with protein particles that had been subjected to an additional drying step (e.g., by vacuum drying) had a significant impact on the initial dispersibility in terms of a more uniform particle size distribution and an overall smaller particle size.

[0233] As shown in Figure 1 (formulations 5a, 5b, 6a, and 6b), Figure 2 (formulations 7a, 7b, 8a, 8b, 9a, 9b, 10a, and 10b), Figure 3 (formulations 11a, 11b, 12a, and 12b), and Figure 4 (formulations 14a, 14b, 15a, and 15b), suspension formulations prepared from exemplary protein particles with residual moisture contents of 3% or greater without an additional vacuum drying step were found to exhibit poor initial dispersibility (e.g., poor uniformity of particle size distribution and generally larger mean (d50) particle diameters) despite the use of either an ultrasonic bath or a high-shear homogenizer for homogenization of the suspensions. Optical microscopy further confirmed the laser diffraction results.

[0234] Example 2 stability studies The suspension formulations prepared according to Example 1 were filled into prefill syringes for N13-2 glass vials (Beva 9a-10b; 0.4 mL) (Macherey-Nagel, Düren, Germany), 2R glass vials (Lys 1a-5d, 11a-11d; mAb:Suc 6a-7d; 1 mL), or Terumo Plajex (mAb:Tre 8a-8d; 1 mL) (Terumo, Tokyo, Japan). To ensure initial injectability, filling was performed manually using B.Braun Injekt (B.Braun AG, Melsungen, Germany) syringes fitted with Terumo Agani 30G (Terumo, Tokyo, Japan) needles (inner diameter ≈160 μm). Vials were closed using 13 mm or 20 mm Teflon-coated injection stoppers and hand-sealed using 10R caps (Westpharma, Exton, USA).

[0235] Particle size stability An increase in particle size of a suspension formulation over time can lead to an increased likelihood of needle clogging for formulations that are to be administered to a subject, for example, by injection, and can also result in changes in release kinetics.

[0236] The particle size stability of suspension formulations containing lysozyme and a model mAb, and containing either sucrose or trehalose as a stabilizer, prepared according to the general method described in Example 1 and stored at 5-8°C, 25°C, and 40°C was investigated.

[0237] Model mAb / sucrose particles suspended in F4H5 and F6H8 Suspension formulations containing model mAb protein particles and sucrose suspended in F6H8 were stored under refrigerated conditions (5-8°C) and at room temperature (25°C) for 6 months. No significant changes in particle size distribution were observed for all tested formulations. Figure 5 shows the particle size distributions of suspension formulations 8a (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from protein particles that had not been vacuum-dried after 6 months of storage at 5°C, 8b (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from protein particles that had been vacuum-dried after 6 months of storage at 5°C, 8a after 6 months of storage at 25°C, and 8b after 6 months of storage at 25°C.

[0238] However, unexpectedly, under higher temperature stress conditions such as 40°C, suspension formulations containing model mAb protein particles and prepared by spray drying and additional vacuum drying, with a residual water content of less than 1.0 wt%, were observed to undergo no significant increase or change in particle size when stored for up to 6 months. In contrast, suspension formulations containing mAb particles that were not subjected to the vacuum drying step during particle preparation were observed to undergo a dramatic increase in particle size upon storage at 40°C for 6 months.

[0239] Figures 6A and 6B show the particle size distribution of a suspension formulation of protein particles containing a model mAb and sucrose in F4H5 as a liquid solvent after storage at 40°C for 0, 1, 3, and 6 months.

[0240] Figure 6A shows the particle size distribution of suspension formulation 7a (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from particles that had not been subjected to vacuum drying and contained a residual water content of approximately 4.2 wt.%. Figure 6B shows the particle size distribution of suspension formulation 7b (50:50 mAb:Suc, TSC = 100 mg / mL) containing a residual water content of approximately 0.1 wt.%.

[0241] Figures 7A and 7B show the particle size distribution of a suspension formulation of protein particles containing a model mAb and sucrose in F6H8 as a liquid solvent after storage at 40°C for 0, 1, 3, and 6 months.

[0242] Figure 7A shows the particle size distribution of suspension formulation 8a (50:50 mAb:Suc, TSC = 100 mg / mL) prepared from particles that had not been subjected to vacuum drying and contained a residual water content of approximately 4.2 wt%. Figure 7B shows the particle size distribution of suspension formulation 8b (50:50 mAb:Suc, TSC = 100 mg / mL) containing a residual water content of approximately 0.1 wt%. Particle size distribution D5 (●), D10 (○), D50 (▼), D90 (△), and D95 ​​(■) values.

[0243] Consistent particle size distributions were observed for both formulations prepared using the spray drying and vacuum drying processes described in Example 1 and using either F4H5 or F6H8 as the liquid solvent (Figures 6B, 7B), which had lower residual moisture content, and stored at 40°C for a period of 6 months, whereas the particle size distribution changed significantly at 6 months for the formulation prepared using protein particles and with higher residual moisture content when no additional drying step was implemented in the process (Figures 6A, 7A).

[0244] SEM analysis of the formulation samples revealed the formation of needle-like structures, indicating that crystallization effects were the underlying cause of the increase in measured particle size. This assumption was further confirmed by XRD analysis of formulation 8a (F6H8, mAb:Suc 50:50; TSC = 100 mg / mL), which showed a peak corresponding to crystalline sucrose (Figure 8, spectrum A). Crystallinity in the XRD spectrum of particles from 8a was not observed after 6 months of storage at 5°C, but was still evident after 6 months of storage at 25°C. On the other hand, a suspension formulation prepared from dried protein-containing particles (formulation 8b) was found to maintain an amorphous state even after 6 months of storage at 40°C (Figure 8, spectrum B).

[0245] Recrystallization of a stabilizer such as sucrose not only has a negative effect on particle size stability, but also on protein stability, as its crystallinity can adversely affect its ability to stabilize the protein. Formulation and storage of protein particles prepared as described above in a non-aqueous liquid solvent such as F4H5 or F6H8, with a water content reduced to less than 1.0 wt. % (e.g., about 0.1 wt. % or less) relative to the weight of the protein particles, can therefore be advantageous in potentially adverse storage conditions, such as when the cold chain or temperature control is not operational.

[0246] It was found that no change in particle size was observed for suspension formulations prepared from spray-dried and vacuum-dried mAb-containing particles and stored in prefillable COP syringes.

[0247] Lysozyme / trehalose in F6H8 The particle size stability of lysozyme / trehalose particles suspended in F6H8 was also investigated. Figures 9A, 9B, and 9C show the particle size stability of formulations prepared with spray-dried and vacuum-dried particles containing different ratios of lysozyme and trehalose suspended in F6H8 as a liquid solvent after storage at 40°C for 0, 1, 3, 6, and 12 months. Figure 9A shows the particle size distribution of Suspension Formulation 2 (Lys:Tre 70:30, TSC = 300 mg / mL). Figure 9B shows the particle size distribution of Suspension Formulation 3 (PS20 containing a 70:30 Lys:Tre formulation, TSC = 100 mg / mL). Figure 9C shows the particle size distribution of Suspension Formulation 4 (Lys:Tre 50:50, TSC = 100 mg / mL).

[0248] As shown in these figures, it was also observed that for protein particles containing lysozyme and trehalose as stabilizers at different concentrations, there was no difference in particle size for these formulations, even after 12 months of storage at 40°C. These results were further confirmed by optical microscopy and SEM. Photographs taken using SEM further showed that there was no change in particle morphology or sintering of individual particles during 12 months of storage at 40°C.

[0249] Resuspension Resuspension, which may also be called redispersibility, of some suspensions prepared according to Example 1 was tested using two different methods.

[0250] The resuspension property of a suspension formulation is another measure of its physical stability. The European Pharmacopoeia (Ph.Eur.) defines general standards for suspensions, which require that the suspension be redispersed by gentle manual shaking. In particular, resuspension of a suspension formulation should not take an excessively long time to provide easy administration by healthcare professionals or even patients themselves. When a suspension formulation is intended to be provided as an administration means, such as in the form of a kit or prefillable syringe, the physical attribute of resuspension property is even more important, as the headspace volume available for resuspending the formulation is generally reduced. An unstable suspension formulation is one that cannot be completely resuspended or redispersed to its original characteristics. For example, a suspension is not stable if it is unable to completely redisperse the particle aggregate structure, including aggregates that may have formed over time under static or storage conditions, as can be seen by visual observation of floaters, sediments, or deposits in the container in which the formulation is stored.

[0251] The first test for resuspension (rotation method) was carried out using an SU1100 vertical rotor (Sunlab, Mannheim, Germany) at a rotation speed of 25 rpm. The time until visual resuspension was achieved was measured. The test was terminated after 15 minutes. The second method (shaking method) was carried out using a Retsch swing mill MM400 (Retsch GmbH, Haan, Germany). For this purpose, the vial containing the suspension was fixed and shaken at a constant frequency for 30 seconds. If no resuspension was observed, this process was repeated at a 2.5 Hz higher frequency (start frequency: 5 Hz; maximum frequency: 30 Hz).

[0252] result Lysozyme / Trehalose Suspension Suspension formulations 1a, 1b, 1c, and 1d containing protein particles, including lysozyme and trehalose particles, were tested for resuspension properties using a vertical shaker (vertical rotation at 25 rpm) stored at 5°C, 25°C, and 40°C. As shown in Figure 10, protein-containing suspension formulations containing F4H5 or F6H8 as the suspension solvent were found to easily resuspend after a few seconds, even for samples stored at 40°C for 12 months. Suspensions based on EO or MCT solvents required longer resuspension times, up to several minutes. Furthermore, in contrast to the semi-fluorinated alkanes, the EO and MCT suspensions were observed to have much lower sedimentation volumes after storage and formed denser cakes.

[0253] Resuspension studies performed on formulations containing higher concentrations of protein particles (2; Lys:Tre 70:30; total solids content (TSC) 300 mg / mL), polysorbate 20 (3; Lys:Tre 70:30; total solids content (TSC) 100 mg / mL; 0.1% polysorbate 20), or higher concentrations of stabilizer (4; Lys:Tre 50:50; 100 mg / mL) in solvent F6H8 also showed that they could be easily redispersed, i.e., in less than 1 minute.

[0254] Lysozyme / sucrose suspension Additionally, suspension formulations containing lysozyme and sucrose particles were tested for resuspension after storage at 5°C, 25°C, and 40°C for 12 months using either the vertical rotation method or the hand shaking method.

[0255] Similar to what was observed for the lysozyme / trehalose particles, it was observed that the 50:50 Lys:Suc particles (TSC=100 gm / mL) in F6H8 (formulations 5a, 5b, 6a, and 6b) were generally more rapidly resuspended using vertical rotation compared to suspensions in EO (Figure 11, graph A). Acceptable redispersion times were observed for the EO-based formulations stored at lower temperatures, while for the F6H8-based formulations, resuspension remained essentially consistent and fast across the various storage temperatures.

[0256] The frequency of vibration required for resuspension for these formulations was also tested when using a shaking method simulating manual shaking to test redispersibility (Figure 11, graph B). Lysozyme suspensions in F6H8 were easily resuspended at a frequency of 5 Hz, the frequency used by operators on average for this procedure. EO formulations required higher frequencies, up to 15 Hz.

[0257] Model mAb / sucrose suspension Resuspension studies were also performed on suspension formulations 7a, 7b, 8a, 8b, 9b, 10b (mAb:Suc 50:50; TSC=100 mg / mL) listed in Table 1 and aged at 5°C, 25°C, and 40°C for a period of 6 months.

[0258] Suspensions containing mAb-containing particles (mAb:sucrose 50:50) (formulations 7a and 8a) prepared without an additional drying step were observed to resuspend less easily than, for example, the lysozyme / sucrose particles described above (Figure 12), and particle scaffold formation was also confirmed. In contrast, however, suspension formulations prepared with mAb:sucrose particles with low residual water content (formulations 7b and 8b) were readily redispersible using the shaking method. Notably, these formulations were also redispersible after prolonged storage at higher temperatures (i.e., 40°C).

[0259] Needle passability and injectability The needle passability of the suspension prepared according to Example 1, i.e., the overall ease with which the formulation can be withdrawn and filled to the volume of the syringe, was tested. The needle passability was tested manually. A 23G needle (Terumo) was attached to a 1 mL B.Braun Inject F disposable syringe (B.Braun AG, Melsungen, Germany). The suspension formulation was then tested for needle passability by moving the plunger toward the end of the syringe. The amount that could be removed was measured.

[0260] Injectability is also an important parameter for suspension formulations intended for administration using a syringe and needle due to the potential risk of needle clogging as a result of particle agglomeration. This can adversely affect the applicability and accurate dosing of the formulation for administration by injection. Syringe gliding force measurements were performed for different injection systems using a Texture Analyzer XT plus (Stable Micro Systems, Godalming, UK). Suspension formulations stored in vials were drawn into 1 mL B.Braun Inject F disposable syringes (B.Braun AG, Melsungen, Germany), which were then fitted with 27G Terumo Agani needles (Terumo, Tokyo, Japan). To determine the gliding force required for injection, the plunger speed was set to obtain a volumetric flow rate of 0.1 mL / sec. To investigate initial dispersion, injectability (27G needle) was manually tested. The 27G needle has an inner diameter of approximately 210 μm.

[0261] result Suspensions prepared using an ultrasonic bath or high-shear homogenizer were generally found to be injectable when suspensions were prepared using spray-dried and vacuum-dried protein-containing particles prepared according to Example 1. In contrast, the use of protein-containing particles that had not been subjected to an additional vacuum drying step resulted in needle clogging as a result of incomplete suspension in some of the formulations tested.

[0262] For example, needle passability tests were performed on suspension formulations 7a, 7b, 8a, and 8b (mAb:Suc 50:50; TSC=100 mg / mL) listed in Table 2 after storage at 40°C for 6 months. It was observed that it was impossible to draw suspensions 7a and 8a into a syringe using a 23G needle. No difficulty was encountered when drawing suspensions 7b and 8b containing mAb particles into a syringe. Even after destroying the particle scaffold at a frequency of 30 Hz, the particles of suspension formulations 7a and 8a were observed to adhere to the vial wall, indicating poor dispersibility. As shown in Table 2, most of the air (measured by the amount that could be removed) was drawn into the syringe.

[0263] JPEG0007761562000002.jpg47128

[0264] Therefore, with regard to additional processing steps, it is believed that including a step of drying (ie, vacuum drying) the protein particles may have a beneficial effect on stability and suitability for use in injection.

[0265] Lysozyme / Trehalose Suspension Following the above protocol, the injectability of suspension formulations containing lysozyme-trehalose-containing particles (lysozyme:trehalose=70:30, TSC=100 mg / mL), formulations 1a, 1b, 1c, and 1d listed in Table 1, stored for 12 months at 40° C. was also tested. For all formulations tested, i.e., in solvents such as F4H5, F6H8, EO, or MCT, no significant changes in gliding force were observed, and no needle clogging was observed, even after storage at 40° C. for 1 year.

[0266] Similar results were observed for lysozyme-trehalose suspension formulations with TSC=300 mg / mL and TSC=100 mg / mL (Formulations 2 and 3 containing 70:30 Lys:Tre, i.e., polysorbate 20). Figure 14 shows the gliding force profiles of Formulations 2 and 3 after 12 months of storage at 40°C.

[0267] Similar results were also obtained with a 50:50 Lys:Tre formulation in F6H8 (Formulation 4, TSC=100 mg / mL).

[0268] Model mAB / sucrose suspension Following the above protocol, the injectability of suspension formulations 7a and 7b (F4H5, 50:50 mAb:Suc; TSC = 100 mg / mL) and 8a and 8b (F6H8, 50:50 mAb:Suc; TSC = 100 mg / mL) containing model mAb-sucrose-containing protein particles, as described in Table 1, stored for 6 months at 40°C, was tested. Figure 15 shows the maximum injection force required for the injection of these formulations stored at 40°C for 6 months. Formulations 7a and 8a, prepared with protein particles that had not been subjected to an additional vacuum drying step, i.e., particles containing a residual moisture content of approximately 4.2 wt%, as shown in Figure 15A, were observed to require increasing application of force as storage at 40°C for 6 months progressed. In contrast, the injectability results for suspension formulations 7b and 8b, which were prepared with protein particles dried under vacuum after spray drying and had a residual moisture content of approximately 0.1 wt. % based on particle weight, appeared to remain constant over a 6-month period (Figure 15B).

[0269] Similar results were obtained for mAb-sucrose suspension formulations prepared with MCT (10b; 50:50 mAb:sucrose, TSC = 100 mg / mL) and EO (9b; 50:50 mAb:sucrose, TSC = 100 mg / mL) as the solvent. Figure 16 shows the gliding force profiles of formulations 9b and 10b after 6 months of storage at 40°C.

[0270] Bevacizumab / sucrose suspension Following the above protocol, the injectability of suspension formulations 14a, 14b (F6H8, 50:50 beva:suc; TSC-100 mg / mL) and 15a, 15b (EO, 50:50 beva:suc; TSC-100 mg / mL) with bevacizumab-sucrose-containing protein particles, as described in Table 1, stored at 40° C. for a period of 6 months, was tested. As shown in FIG. 17, the bevacizumab-containing suspension formulations were found to be injectable without any needle clogging or failure after 6 months of storage in both F6H8 and EO.

[0271] Protein Activity Test An ELISA was used to evaluate the activity of bevacizumab in formulation 16. Bevacizumab is involved in binding to VEGF, which is associated with the inhibition of angiogenesis. The test is based on a sandwich ELISA using microtiter plates coated with recombinant human VEGF-A. Bound bevacizumab was quantified using a horseradish peroxidase (HRP)-conjugated anti-human IgG monoclonal antibody, which binds to the Fc region of the antibody. The ELISA assay was performed using a commercially available kit from ImmunoGuide (Ankara, Turkey) according to the manufacturer's instructions, utilizing aqueous commercial protein raw material and reconstituted protein suspension formulations.

[0272] A protein suspension formulation was prepared from the spray-dried and then vacuum-dried protein particles. ELISA analysis showed no significant difference in binding activity between the raw material and the reconstituted protein suspension formulation. The processes of protein particle preparation, subsequent drying, and subsequent drying of the suspension did not adversely affect the activity of the anti-VEGF protein bevacizumab.

[0273] JPEG0007761562000003.jpg41129

Claims

1. 1. A method for producing a suspension formulation comprising protein particles and a non-aqueous solvent, the method comprising: a) providing an aqueous solution comprising a protein and a stabilizing agent; b) removing water from the aqueous solution to obtain solid protein particles; c) further drying the protein particles obtained in step b) to obtain protein particles containing a residual water content of less than 0.5 wt. %, based on the weight of the particles; and d) suspending the protein particles of step c) in a non-aqueous solvent comprising a semi-fluorinated alkane selected from F(CF2)4(CH2)5H or F(CF2)6(CH2)8H, and optionally e) homogenizing the suspension; The method, wherein the protein particles comprise a protein and a stabilizing agent, the stabilizing agent is a sugar, and the non-aqueous solvent comprises a semi-fluorinated alkane.

2. 10. The method of claim 1, wherein the water of step b) is removed by spray drying or freeze drying the composition.

3. 3. The method according to claim 1, wherein the stabilizing agent is a sugar selected from trehalose and sucrose.

4. The method according to any one of claims 1 to 3, wherein the protein has a molecular mass of 3 to 200 kDa.

5. 5. The method according to any one of claims 1 to 4, wherein after step d) or optionally step e), at least 90% of the protein particles have an average particle size of 1 to 30 μm as determined by laser diffraction.

6. The method of any one of claims 1 to 5, wherein step e) comprises high shear homogenization, milling or sonication.

7. A suspension formulation comprising protein particles suspended in a non-aqueous solvent and obtainable by the method of any one of claims 1 to 6, wherein the protein particles comprise a protein and a stabilizer, and the non-aqueous solvent comprises a semi-fluorinated alkane selected from F(CF2)4(CH2)5H or F(CF2)6(CH2)8H.

8. A suspension formulation comprising protein particles comprising a protein and a stabilizer, suspended in a non-aqueous liquid solvent comprising a semi-fluorinated alkane selected from F(CF 2 ) 4 (CH 2 ) 5 H or F(CF 2 ) 6 (CH 2 ) 8 H, and dried twice, wherein the residual water content of the protein particles is less than 0.5% by weight, the stabilizer is a sugar, and at least 90% of the protein particles have an average particle size of 1 to 30 μm as determined by laser diffraction.

9. 9. The suspension formulation of claim 8, wherein the protein has a molecular mass of 3 to 200 kDa.

10. The suspension formulation of any one of claims 8 to 9, wherein the protein is selected from an antigen-binding polypeptide or protein, a vaccine, and an enzyme.

11. 11. The suspension formulation of any one of claims 8 to 10, wherein the relative weight ratio of protein to stabiliser is in the range of 1:1 to 7:

3.

12. 12. The suspension formulation of any one of claims 8 to 11, wherein the residual water content of the suspension formulation is less than 1.0 mg / mL, based on the total volume of the formulation.

13. A kit comprising a suspension formulation as defined in any one of claims 7 to 12, and a container adapted to hold said formulation, and optionally dispensing means.

14. 13. A dispensing device comprising a suspension formulation as defined in any one of claims 7 to 12, the dispensing device being adapted to administer said suspension formulation by topical administration or injection.

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