IgG:tgfβrii fusion protein compositions

Abstract

This invention relates to pharmaceutical compositions, particularly pharmaceutical compositions comprising an IgG:TGFβRII (e.g., an anti-PD-L1:TGFβ inhibitor) fusion protein. The invention also particularly relates to methods for manufacturing said compositions, kits comprising said compositions, packaging comprising said compositions, methods for manufacturing said packaging, and treatment methods using said compositions and / or packaging, particularly for cancer treatment.

Description

Technical Field

[0001] This invention relates to pharmaceutical compositions, specifically pharmaceutical compositions comprising IgG:TGFβRII fusion protein. The invention also particularly relates to methods for manufacturing the composition, kits comprising the composition, containers or drug delivery devices comprising the composition, methods for manufacturing the container or drug delivery device, and methods for treatment (especially cancer treatment) using the composition and / or the container or drug delivery device. Background Technology

[0002] WO2015118175 describes a bifunctional IgG:TGFβRII fusion protein that combines an anti-programmed death-ligand 1 (anti-PD-L1) antibody with the soluble extracellular domain of tumor growth factor β receptor type II (TGFβRII) into a TGFβ-neutralizing "trap" in a single molecule. Specifically, this protein is a heterotetramer composed of two immunoglobulin light chains and two heavy chains of the anti-PD-L1 antibody. The two heavy chains contain an anti-PD-L1 antibody heavy chain fused to the extracellular domain of human TGFβRII via a flexible glycine-serine linker gene (see Figure 1). This anti-PD-L1 / TGFβ trap molecule is designed to target two major immunosuppressive mechanisms in the tumor microenvironment, and therefore could be used to treat cancer or inhibit tumor growth.

[0003] One object of the present invention is to provide a viable pharmaceutical composition of an IgG:TGFβRII fusion protein. In the field of formulation of biologics, particularly those containing antibodies or antibody fragments, unpredictability is inherent, making it difficult to discover such viable pharmaceutical compositions because most formulations of a given biologic (if the formulation is arbitrarily chosen) are unstable over long periods and / or under stress conditions, due to multiple degradation pathways open to biologics (especially aqueous formulations). For example, degradation factors may include one or more of the following (typically two or more, possibly three or more):

[0004] ●Physical effects, for example:

[0005] ○ Insufficient inhibition of the aggregation of related protein molecules;

[0006] ○ Insufficient inhibition of precipitation;

[0007] ○ Insufficient inhibition of the adsorption of related protein molecules at the water-air interface or the contact surface of any packaging material;

[0008] ○ Insufficient osmotic pressure regulation;

[0009] ● Chemical reactions, for example:

[0010] Insufficient oxidation regulation;

[0011] Insufficient inhibition of photo-oxidation;

[0012] Insufficient inhibition of ester bond hydrolysis leads to the formation of acid, aldehyde and peroxide products, thereby affecting the stability of the fusion protein;

[0013] ○ Insufficient pH stability and maintenance;

[0014] ○ Insufficient inhibition of protein fragmentation;

[0015] ○ Insufficient inhibition of protein unfolding.

[0016] Any, some, or all of the above factors may result in an unworkable medicine (whose use in medical treatment may be unsafe) or a medicine with variable and unpredictable activity, especially considering variable pressures (stirring, freeze-thaw, heating, light exposure), and the fact that different batches of medicine may be exposed to the environment during production, transportation, and storage.

[0017] The present invention preferably seeks to solve one or more of the above-mentioned stability problems, and thereby provides a feasible pharmaceutical formulation. Summary of the Invention

[0018] In some embodiments, pharmaceutical compositions comprising the IgG:TGFβR fusion protein are provided. The pharmaceutical compositions of the present invention may preferably comprise, consist of, or exclude any, some, or all of the components described herein (e.g., any one of buffers, surfactants, sugar components, amino acid components, tonicators, antioxidants, chelating agents; or virtually exclude any of the foregoing), present in any relevant amounts described herein, and / or may preferably be characterized by any, some, or all of the parameters described herein (e.g., pH, pI, osmolality). The pharmaceutical composition may be a liquid (e.g., aqueous) pharmaceutical composition. Alternatively, the pharmaceutical composition may be a lyophilized composition.

[0019] In some embodiments, a container or drug delivery device is provided that contains or includes a pharmaceutical composition as defined herein. Such a drug delivery device may be, for example, a vial, ampoule, syringe, pre-filled syringe, injection pen (e.g., substantially incorporated into a syringe), autoinjector, or intravenous infusion bag, or a package / container containing any of the foregoing.

[0020] In some embodiments, a component kit is provided, which includes a drug delivery device, a pharmaceutical composition as defined herein, and optionally a set of instructions for administration of the pharmaceutical composition (e.g., intravenous, subcutaneous).

[0021] In some embodiments, a method for preparing a pharmaceutical composition is provided, the method comprising mixing an IgG:TGFβR fusion protein with one or more pharmaceutically acceptable excipients and / or carriers.

[0022] In some embodiments, a method is provided for treating a disease or medical condition in a patient who requires such treatment, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0023] In some embodiments, pharmaceutical compositions as defined herein are provided for treating diseases or medical conditions in patients who require such treatment.

[0024] In some embodiments, the use of the pharmaceutical compositions defined herein in the preparation of a medicament for treating a disease or condition is provided.

[0025] In some embodiments, methods for treating a disease or medical condition, pharmaceutical compositions for treating a disease or medical condition are provided, and the use of the pharmaceutical composition in the preparation of a medicament for treating a disease or medical condition as defined herein, wherein the disease or medical condition is a PD-L1-related disease, a TGFβ-related disease, and / or a proliferative disease or condition, preferably cancer.

[0026] In some embodiments, the treatment or therapy involves combination therapy, wherein the pharmaceutical composition is administered in combination with one or more other pharmaceutical or bioactive substances; wherein the combination therapy involves simultaneous, sequential, or separate administration of the various components of the treatment. In some embodiments, the additional pharmaceutical or bioactive substance may be present in any pharmaceutical composition as defined herein.

[0027] All of the above-described treatment methods, applications of one or more compositions, and uses of one or more compositions in the manufacture of pharmaceuticals are equally applicable to related containers, drug delivery devices, and kits containing one or more of the aforementioned compositions.

[0028] Any feature (including optional, suitable, and preferred features) relating to any specific aspect of the invention may also be a feature (including optional, suitable, and preferred features) relating to other aspects of the invention, unless incompatible therewith.

[0029] Brief description of the attached figures

[0030] To better understand the present invention and to illustrate how embodiments of the present invention can operate effectively, reference is made to the following figures, in which:

[0031] Figure 1AThe sequence of half of the bintrafusp alfa fusion protein is shown, which contains a light chain (with designated VL and CL regions) and a heavy chain (with designated VH, CH1, CH2 and CH3 regions) that are linked to the extracellular domain (ECD) of TGFβ receptor II via designated linkers.

[0032] Figure 1B The sequence of half of the bintrafusp alfa fusion protein is shown, comprising a light chain (with designated VL and CL regions) and a heavy chain (with designated VH, CH1, CH2, and CH3 regions) connected to the extracellular domain (ECD) of TGFβ receptor II via a designated linker. The overall sequences of the individual light chains, heavy chains, linkers, and extracellular domains of TGFβ receptor II are shown for... Figure 1A and Figure 1B They are all the same, and Figure 1A and Figure 1B In terms of the overall sequence, it is intended to correspond to “bintrafusp alfa”, as defined in the definition section of this application, with reference to CAS Registry No. 1918149-01-5. Figure 1B and Figure 1A Same, except: Figure 1A The first three amino acids (PCPs) in the CH2 region are Figure 1B The last three amino acids (PCP) of the hinge region; and, Figure 1A The last three amino acids (GQP) of the VL region are Figure 1B The first three amino acids (GQP) in the CL region. Figure 1A and Figure 1B The specific sequence allocation for the specific regions shown is for ease of comparison between them. For region-specific comparison purposes, methods such as... Figure 1A or Figure 1B The specific sequence shown is allocated to a specific region. In some implementations, it can be used... Figure 1B The specific sequence is assigned to a specific region.

[0033] Figure 2 The graph shows the relationship between Tg' (in °C) and NaCl concentration for formulations containing 8% trehalose (rhombus), 4% trehalose (square), and 2% trehalose (triangle). From a practical standpoint during lyophilization, a Tg' value below -40 °C is undesirable.

[0034] Figure 3 It is a contour plot where the Tm3 unfolding temperature is represented as contour lines (range from 69.98 to 72.72) within a formulation space (with fixed ionic strength, 95 mM NaCl) with variable protein concentration (mg / mL bintrafusp alfa) and pH.

[0035] Figure 4 It is a contour plot where the Tm3 unfolding temperature is represented as contours (ranging from 69.98 to 72.72) within a formulation space (with a fixed pH of pH 6.25) having variable protein concentrations (mg / mL bintrafusp alfa) and ionic strengths (mM NaCl).

[0036] Figure 5 This is a contour plot where %LMW of the substance after 4 weeks at 40°C is represented by contour lines (range 5.71–12.35) within a formulation space (with fixed ionic strength, 95 mM NaCl) with variable protein concentration (mg / mL bintrafusp alfa) and pH. Data were obtained via CGE NR (i.e., CGE under non-reducing conditions).

[0037] Figure 6 This is a contour plot where %LMW of the substance after 4 weeks at 40°C is represented as contours within a formulation space (with a fixed pH of 6.25) with variable protein concentrations (mg / mL bintrafusp alfa) and ionic strengths (mM NaCl) (see contours at 6, 6.5, 7, and 7.5). Data obtained by CGE NR.

[0038] Figure 7 This is a contour plot where %HMW of matter after 4 weeks at 40°C is represented by contours (range 1.2–6.3) within a formulation space (with fixed ionic strength, 95 mM NaCl) with variable protein concentration (mg / mL bintrafusp alfa) and pH. Data were obtained by SE-UPLC.

[0039] Figure 8 This is a contour plot where %HMW of matter after 4 weeks at 40°C is represented as contours within a formulation space (with a fixed pH of pH 6.25) with variable protein concentration (mg / mL bintrafusp alfa) and ionic strength (mM NaCl) (see contours at 1, 2, 3, and 4). Data were obtained by SE-UPLC.

[0040] Figure 9 This is a contour plot, where the Cluster 2 isotypes after 4 weeks at 40°C are represented by contours (range 5.5–13.68) within a formulation space with variable protein concentrations (mg / mL bintrafusp alfa) and pH (with fixed ionic strength, 95 mM NaCl). Data were obtained through cIEF identification and isotype distribution of iCE3.

[0041] Figure 10 This is a contour plot where the % oxidation level of Met516 after 4 weeks at 40°C is represented by contour lines (range 5.77–8.76) within a formulation space (with fixed ionic strength, 95 mM NaCl) with variable protein concentration (mg / mL bintrafusp alfa) and pH. Data were obtained by RP-UPLC.

[0042] Figure 11 This is a contour plot where the % oxidation of Met516 after 4 weeks at 40°C is represented by contours within a formulation space (with a fixed pH of pH 6.25) with variable protein concentration (mg / mL bintrafusp alfa) and ionic strength (mM NaCl) (see contours at 6, 6.2, 6.4, and 6.6). Data were obtained by RP-UPLC.

[0043] Figure 12 The scatter plot shows the relationship between % oxidation and bintrafusp alfa concentration after photopressure.

[0044] Figure 13 This is a scatter plot of all samples, showing the relationship between % oxidation and ionic intensity after photopressure.

[0045] Figure 14 This is a scatter plot of all samples, showing the relationship between % oxidation and pH after photopressure.

[0046] Figure 15 These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contours (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration of 20 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions at this IgG:TGFβR2 concentration are pH 5.7 and an ionic strength of 40 mM NaCl.

[0047] Figure 16 These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contours (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration of 40 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions at this IgG:TGFβR2 concentration are pH 5.9 and an ionic strength of 60 mM NaCl.

[0048] Figure 17These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contours (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration of 60 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions at this IgG:TGFβR2 concentration are pH 5.9 and an ionic strength of 150 mM NaCl.

[0049] Figure 18 This is a graph showing how %HMW changes with protein concentration after 4 weeks of heat stress at 40°C when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for a specific protein concentration accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0050] Figure 19 This is a graph showing how %HMW changes with protein concentration after 8 weeks of heat stress at 40°C when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for a specific protein concentration accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0051] Figure 20 This is a graph showing how the main clipping changes with protein concentration after 4 weeks of heat stress at 40°C when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for a specific protein concentration accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0052] Figure 21 This is a graph showing how %LMW changes with “different excipients” after 4 weeks of heat stress at 40°C when the surfactant is fixed as polysorbate 20 and the ionic strength is fixed at any NaCl concentration applicable to the specific protein concentrations of polysorbate 20 listed in Table 3J. Measured values ​​are represented by circles, while predicted values ​​based on the measured values ​​are represented by squares.

[0053] Figure 22 This is a graph showing how the percentage of deamidation forms changes with protein concentration after 4 weeks at 40°C when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0054] Figure 23 This is a graph showing how the percentage of oxidized form changes with excipients for surfactants polysorbate 20 (squares indicate predicted values) and Kolliphor 188 (triangles indicate predicted values ​​based on measured values, represented by circles), when the protein (bintrafusp alfa) concentration is fixed at 40 mg / mL, after 4 weeks at 40°C.

[0055] Figure 24 This is a graph showing how the percentage of oxidized form of protein (bintrafusp alfa) changes with the excipient after 8 weeks at 40°C when the protein concentration is fixed at 40 mg / mL.

[0056] Figure 25 This graph shows how %cluster 1 (via iCE3) changes with excipients after 4 weeks of thermostressing at 40°C when the protein (bintrafusp alfa) concentration is fixed at 40 mg / mL. Circles represent measured values, and squares represent predicted values ​​based on the measured values.

[0057] Figure 26 This is a graph showing how % cluster 2 (via iCE3) changes with protein concentration after 4 weeks at 40°C when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0058] Figure 27 This is a graph showing how %NMW (by SE-UPLC) changes with protein concentration after photopressure when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measurement, indicated by circles in the graph).

[0059] Figure 28 This is a graph showing how the main clipping wave (via CGE) changes with protein concentration after photopressure when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0060] Figure 29This is a graph showing how %LMW (by CGE) changes with protein concentration after photopressure when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measurement, indicated by circles in the graph).

[0061] Figure 30 This is a graph showing how the percentage of oxidized form (by RO-UPLC) changes with protein concentration after photopressure when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0062] Figure 31 This is a graph showing how %HMW changes with protein concentration after 3 freeze-thaw cycles when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is the value used in Table 3J for the specific protein concentrations accompanied by polysorbate 20 and the “various excipients” shown in the legend (the legend refers to the predicted value based on the measured value, indicated by circles in the graph).

[0063] Figure 32 It is a bar chart showing how %HMW (by SE-UPLC) changed with formulation (corresponding to the formulation number in Table 3J, given on the horizontal axis) before and after 3 days of mechanical pressure (300 rpm).

[0064] Figure 33 It is a graph showing how %HMW (after mechanical pressure) changes with surfactant when all other factors are averaged.

[0065] Figure 34 This is a graph showing how overall desirability (based on a balance of multiple factors, stress tests, and results) varies with excipients for protein (bintrafusp alfa) concentrations of 40 mg / mL (predicted values ​​shown as squares, based on measurements shown as circles), 50 mg / mL (predicted values ​​shown as triangles, based on measurements shown as circles), and 60 mg / mL (predicted values ​​shown as rhombuses, based on measurements shown as circles).

[0066] Figure 35This is a graph showing how overall desirability (based on a balance of multiple factors, stress tests, and results) varies with excipients for the surfactants polysorbate 20 (shown as predicted values, based on measurements shown as circles) and Kolliphor 188 (shown as predicted values, based on measurements shown as circles) when the protein concentration is fixed at 40 mg / mL.

[0067] Figure 36 The bar chart shows the increase in % HMW after heat stress (by SE-UPLC) for each formulation in Table 3K and Table 2A 2A.5 (2A.5) (referred to as “01-300518”).

[0068] Figure 37 The bar chart shows the % LMW increase after heat stress (by CGE-NRED) for each formulation in Table 3K and Table 2A.5 (referred to as “01-300518”).

[0069] Figure 38 The bar charts show the percentage oxidation after thermal stress (by RP-UPLC) for each formulation in Table 3K and Table 2A.5 (referred to as “01-300518”).

[0070] Figure 39 The bar chart shows the percentage of deamidation (by IEX) after heat stress for each formulation in Table 3K and Table 2A.5 (referred to as “01-300518”).

[0071] Figure 40 The bar chart shows the percentage purity (by CGE-RED, i.e., CGE under reducing conditions) of each formulation after heat stress for Table 3K and Table 2A.5 (referred to as “01-300518”).

[0072] Figure 41 The bar chart shows the % main clipping (by CGE-RED) after heat stress for each formulation in Table 3K and Table 2A.5 (referred to as “01-300518”).

[0073] Figure 42 The bar chart shows the % HMW increase of each formulation in Table 3K after photopressure (by SE-UPLC).

[0074] Figure 43 The bar chart shows the percentage oxidation of each formulation of Table 3K after photopressure (by RP-UPLC).

[0075] Figure 44 The bar chart shows the % LMW (by CGE-NRED) of each formulation of Table 3K after photopressure.

[0076] Figure 45 The bar chart shows the % purity (by CGE-RED) of each formulation of Table 3K after photopressure.

[0077] Figure 46 The bar chart shows the % main clipping (by CGE-RED) of each formulation in Table 3K after photopressure.

[0078] Figure 47 The bar chart shows the % HMW of each formulation of Table 3K after mechanical stress (by SE-UPLC).

[0079] Figure 48 The bar chart shows the % LMW (by CGE-NRED) of each formulation of Table 3K after mechanical stress.

[0080] Figure 49 The bar chart shows the % purity (by CGE-RED) of each formulation of Table 3K after mechanical pressure.

[0081] Figure 50 The bar chart shows the % main clipping wave (by CGE-RED) of each formulation in Table 3K after mechanical pressure.

[0082] Figure 51 Display a time-temperature curve to illustrate how to perform 3 FT cycles.

[0083] Figure 52 It is a bar chart showing how the pH of all formulations F1-F20 changed at different time points during the pressure test, including (the bars for each formulation listed from left to right): time = 0; after 3 FT cycles; after 2 weeks at 25°C, after 2 weeks at 40°C, after 4 weeks at 25°C, and after 4 weeks at 40°C.

[0084] Figure 53 It is a bar chart showing the weight permeable molar concentration of each of the formulations F1-F20.

[0085] Figure 54 It is a bar chart showing how the turbidity of all formulations F1-F20 changed at different time points during the pressure test, including (the bars for each formulation listed from left to right): time = 0; after 3 FT cycles; after 2 weeks at 25°C, after 2 weeks at 40°C, after 4 weeks at 25°C, and after 4 weeks at 40°C.

[0086] Figure 55 It is a bar graph showing how the temperature of the Tm2 peak of the nano-DSC traces of all formulations F1-F20 changes at different time points during the pressure test, including (the bars for each formulation listed from left to right): time = 0; after 3 FT cycles; 2 weeks at 25°C, 2 weeks at 40°C, 4 weeks at 25°C, and 4 weeks at 40°C.

[0087] Figure 56 It is a bar graph showing how the onset temperature Tm of the nano-DSC traces of all formulations F1-F20 changes at different time points during the pressure test, including (the bars for each formulation are listed from left to right): time = 0; after 3 FT cycles; 2 weeks at 25°C, 2 weeks at 40°C, 4 weeks at 25°C, and 4 weeks at 40°C.

[0088] Figure 57 is a bar chart showing how %HMW (by SE-UPLC) varies with formulation (the formulation numbers corresponding to those in Table 5A are given on the horizontal axis). (A), (C), and (E) show %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the differences in %HMW after 4 weeks at time = 0 and 40°C.

[0089] Figure 58 is a bar chart showing how %LMW (by CGE-NRED) changes with formulation (the formulation numbers corresponding to those in Table 5A are given on the horizontal axis). (A), (C), and (E) show %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the differences in %LMW after 4 weeks at time = 0 and 40°C.

[0090] Figure 59 It is a 3D contour plot with a desirability parameter (reflecting the balance of factors in the overall response assessment), which is represented as a surface in a formulation space with variable trehalose concentration and ionic strength (given in mM NaCl) and a fixed arginine concentration.

[0091] Figure 60 It is a 3D contour plot with a desirability parameter (reflecting the balance of factors in the overall response assessment), which is represented as a surface in a formulation space with variable arginine concentration and ionic strength (given in mM NaCl) and a fixed trehalose concentration.

[0092] Figure 61 It is a 3D contour plot with a desirability parameter (reflecting the balance of factors in the overall response assessment), which is represented as a surface in the formulation space with variable arginine and trehalose concentrations and a fixed ionic strength concentration (given in mM NaCl). Detailed Implementation

[0093] definition

[0094] Unless otherwise stated, the following terms used in the specification and claims shall have the following meanings.

[0095] Throughout the description and claims of this specification, the words “comprising” and “including” and variations thereof mean “including, but not limited to”, and they are not intended to exclude other parts, additives, quantities, integers, or steps. Throughout the description and claims of this specification, unless the context requires otherwise, the singular includes the plural. In particular, where the indefinite article is used, unless the context requires otherwise, the specification shall be understood to be in consideration of both the plural and the singular.

[0096] In this document, when referring to "antibody," such as IgG (e.g., when using the symbol, such as IgG:TGFβR fusion protein), it preferably refers to an intact antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment preferably comprises at least an Fv region or ScFv, more preferably at least a Fab region or an F(ab)2 fragment. However, in a more preferred embodiment, when referring to "antibody," such as IgG (or any subtype thereof, such as IgG1, IgG4), it refers to an intact antibody.

[0097] A complete antibody typically contains two fragment antigen-binding (Fab) regions (or Fab domains) in its monomeric form, optionally labeled F(ab)2, usually linked by a hinge region to a crystallizable fragment (Fc) region (or Fc domain). This structure consists of four polypeptide chains—two identical heavy chains and two identical light chains, all interconnected by disulfide bonds. Each Fab domain contains a single heavy chain (e.g., V) linked by disulfide bonds. H -C H 1) and single light chains (e.g., V) L -C L The pairing of parts of the Fc domain. The Fc domain contains the two remaining parts of the heavy chain (e.g., C). H 2-C H 3) Pairing.

[0098] Complete IgG is a monomeric antibody and has the structure described above for complete antibodies. In the case of complete IgG, each identical heavy chain contains a variable heavy chain region (V0) in sequence from the N-terminus to the C-terminus. H ), first constant heavy chain region (C H 1), the second constant heavy chain region (C H 2) and the third constant heavy chain region (C H 3). Simultaneously, each identical light chain contains a variable light chain region (V) in order from the N end to the C end. L ) and constant light chain region ( CL V of the heavy chain H -C H Part 1 and light chain V L -C LLinked together by disulfide bonds, they form one of the pair of Fab regions of a complete IgG. The two identical C-cells of the heavy chain are linked together by disulfide bonds. H 2-C H The three parts form the Fc region of complete IgG. Complete IgG has a molecular weight between 140 kDa and 180 kDa, preferably 140-160 kDa (more preferably 144-155 kDa, most preferably about 146 kDa) or 165-175 kDa (more preferably 168-172 kDa, most preferably about 170 kDa). Complete IgG typically has a pI of 6 to 9.5.

[0099] Intact IgG antibodies have four distinct subclasses: IgG1, IgG2, IgG3, and IgG4. All subclasses share the same core region as described above, but differ slightly, particularly in the number of amino acids and / or disulfide bonds in the hinge region. Structurally, IgG1 and IgG4 are considered the most similar. Intact IgG1, IgG2, or IgG4 typically have a molecular weight of 140-160 kDa (more preferably 144-155 kDa, most preferably about 146 kDa), while intact IgG3 typically has a molecular weight of 165-175 kDa (more preferably 168-172 kDa, most preferably about 170 kDa). Typically, the pI of intact IgG1 is 8-9.4, more preferably 8.2-9.2. Typically, the pI of intact IgG2 is 6.5-8.5, more preferably 7.0-8.0. Typically, the pI of intact IgG3 is 7-9.5, more preferably 7.5-9.0. Typically, the pI of intact IgG4 is 6-8.5, more preferably 6.4-8.

[0100] For example, antigen-binding fragments may include: Fab region (e.g., its corresponding light chain V). L -C L Paired V H -C H 1) or F(ab)2 region with two connected Fab regions; Fv region (e.g., its corresponding variable light chain portion V) L Paired V H ); and / or, single-chain variable fragment ScFv domains (e.g., via peptide linkers with V L Connected V H (Preferably containing about 25 amino acids). For the purposes of this invention, when referring to IgG, such as in IgG:TGFβR fusion proteins (including more specifically defined fusion proteins, such as anti-PD-L1(IgG):TGFβR2 fusion proteins), it can be complete IgG, for example, fused to TGFβR2, or an antigen-binding fragment (such as those described above), for example, fused to TGFβR2. However, most preferably, complete IgG is preferred in such fusion proteins.

[0101] The terms "TGF-β receptor" (TGFβR), "TGF-β receptor I" (TGFβR1), or "TGF-β receptor II" (TGFβR2) are well known in the art. For the purposes of this disclosure, these receptors include complete receptors and fragments capable of binding TGF-β. Preferably, it is an extracellular domain of the receptor or an extracellular domain fragment capable of binding TGF-β.

[0102] The term "fusion protein" is well known in the art. IgG:TGFβR fusion protein is an IgG antibody (preferably a monoclonal antibody, preferably in homodimeric form) fused to the TGF-β receptor. The nomenclature of anti-PD-L1(IgG1):TGFβR2 fusion protein indicates an anti-PD-L1 IgG1 antibody fused to TGF-β receptor II (preferably a fragment of its extracellular domain capable of binding TGF-β).

[0103] “Bintrafusp alfa” is well known in the art. Bintrafusp alfa is an anti-PD-L1 (IgG1):TGFβR2 fusion protein, described in CAS Registry No. 1918149-01-5. It is also described in WO 2015 / 118175, Lan et al. (“Enhanced preclinical antitumor activity of M7824, abifunctional fusion protein simultaneously targeting PD-L1 and TGF-β”, Sci. Transl. Med. 10, 2018, pp. 1-15). Specifically, bintrafusp alfa is a fully human immunoglobulin G1 (IgG1) monoclonal antibody against human PD-L1, fused to the extracellular domain of human TGFβ receptor II (TGFβR2). Therefore, bintrafusp alfa is a bifunctional fusion protein that simultaneously targets the PD-L1 and TGFβ pathways. Specifically, Example 1 on page 34 of WO 2015118175 describes bintrafusp alfa as follows (bintrafusp alfa is referred to therein as "anti-PD-L1 / TGFβ trap"):

[0104] The term "anti-PD-L1 / TGFβ trap" refers to an anti-PD-L1 antibody-TGFβ receptor II fusion protein. The light chain of this molecule is identical to the light chain of the anti-PD-L1 antibody (SEQ ID NO:1). The heavy chain of this molecule (SEQ ID NO:3) is a fusion protein in which the heavy chain of the anti-PD-L1 antibody (SEQ ID NO:2) is fused to the N-terminus of a soluble TGFβ receptor II (SEQ ID NO:10) via a flexible (Gly4Ser)4Gly linker (SEQ ID NO:11). At the fusion junction, the C-terminal lysine residue of the antibody heavy chain is mutated to alanine, thereby reducing protease cleavage.

[0105] For purposes of this disclosure and molar calculations, the molecular weight (Mw) of bintrafusp alfa is considered to be 182 kilodaltons (kDa), or 182,000 g / mol. Therefore, a liquid pharmaceutical composition containing 10 mg / mL bintrafusp alfa can be considered as its 0.055 mM solution, a liquid pharmaceutical composition containing 40 mg / mL bintrafusp alfa can be considered as its 0.220 mM solution, and a liquid pharmaceutical composition containing 50 mg / mL bintrafusp alfa can be considered as its 0.275 mM solution.

[0106] Any specific fusion protein mentioned herein, including any IgG:TGFβR fusion protein described herein (especially bintrafusp alfa), including the associated original pharmaceutical substances, whether they are commercially available, described in patent documents, or as described elsewhere in the art, and their biosimilars.

[0107] This document, by reference to any specific IgG:TGFβR fusion protein as defined herein (especially IgG:TGFβR2 fusion protein, more specifically IgG:TGFβR2 fusion protein having the amino acid sequence of bintrafusp alfa), may include variants that (1) have at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence disclosed with respect to said IgG:TGFβR fusion protein, or (2) have no more than 30%, no more than 20%, no more than 10%, or no more than 5% amino acid residue difference with those disclosed with respect to said IgG:TGFβR fusion protein. Preferably, any specific IgG:TGFβR fusion protein referred to herein by reference to the nucleotide or amino acid sequence described herein (especially IgG:TGFβR2 fusion protein, more specifically IgG:TGFβR2 fusion protein having the amino acid sequence of bintrafusp alfa) refers to a protein having said exact sequence (and not any variant thereof).

[0108] The term sequence identity is well known in the art. Sequence identity can be determined using the Needleman-Wunsch algorithm for global alignment of two sequences (Needleman and Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, J Mol Biol 48(3), 443-53, March 1970).

[0109] The indication that a protein has a certain amount of amino acid residue “difference” means that, compared with a reference sequence, that amount of residues has been inserted, deleted and / or substituted.

[0110] In this document, the terms "buffer solution," "buffer system," or "buffer solution" refer to an aqueous solution typically containing a mixture of acids (usually weak acids, such as acetic acid, citric acid, or histidine in the form of imidazoline), and their conjugate bases (e.g., acetates or citrates, such as sodium acetate, sodium citrate, or histidine) or a base (usually a weak base, such as histidine) and its conjugate acid (e.g., protonated histidine). Due to the "buffering effect" provided by the buffer system, the pH of a "buffer solution" changes only slightly upon the addition of a small amount of a strong acid or base. A buffer solution may contain one or more buffer systems, but preferably no more than two (i.e., a dual buffer system, such as histidine-citrate, histidine-acetate, or citrate-phosphate), but most preferably, the buffer solution contains one and at most one buffer system.

[0111] Here, in the context of this specification, "strong acid" is preferably a pK of -1.0 or lower. a Acids, and "weak acids" are preferably those with a pK value of 2.0 or greater. a Acids. Here, in the context of this specification, a "strong base" is preferably the pK of its conjugate acid. a The base is 12 or higher (preferably 14 or higher), and a "weak base" is preferably the base whose conjugate acid has a pK value of 12 or higher. a A base of 10 or lower.

[0112] Unless otherwise stated, the "pK" mentioned in this article refers to... a "This should be interpreted as pK in water under standard ambient temperature and pressure (SATP)." a Value, preferably the conjugate acid of the relevant substances.

[0113] An "amino acid component" is one or more components containing one or more amino acids, and an amino acid component can be composed of a single amino acid.

[0114] Unless otherwise stated, references herein to one or more "amino acids," whether specific (e.g., arginine, histidine) or generic (e.g., any amino acid), refer to the corresponding amino acid in the presence or otherwise of them in the composition (especially the pharmaceutical liquid composition of the present invention). Free amino acids (Regardless of their protonation state and / or salt form, however for consistency, the amount is preferably calculated by referring to the free amino acid itself.) This may preferably include natural and / or artificial amino acids. Unless otherwise stated, these references are not intended to refer to as a larger compound (unlike compositions comprising multiple compounds). Part covalent integration amino acid residuesExamples of proteins include peptides or proteins (where such amino acid residues are linked by peptide bonds). Therefore, although an antibody in protein form contains amino acid residues, it is not considered to contain any "free amino acids." For example, a composition defined as "arginine-free" does not contain any free arginine, but it may still contain one or more proteins that themselves contain arginine residues (e.g., bintrafusp alfa).

[0115] Unless otherwise stated, any one or more “amino acids” mentioned herein, whether specifically or generally, preferably refer to the L-stereomer or its racemic mixture, and most preferably L-amino acids.

[0116] In this article, "sugar component" refers to one or more components, including one or more sugars and / or sugar alcohols, although a sugar component may consist of a single sugar or sugar alcohol.

[0117] In this article, “non-reducing sugar” is a sugar that has no aldehyde moiety or no ability to form an aldehyde moiety (e.g., through isomerism).

[0118] In this document, "tension modifier" or "tension regulator" refers to a reagent contained in a composition that contributes to (or increases) the total weight osmotic molar concentration and volumetric osmotic molar concentration of the composition. Preferably, as used herein, a tension modifier includes a reagent that serves to make the solution osmotically similar to that of a physiological fluid.

[0119] In this document, "antioxidant" or "antioxidant component" refers to one or more components comprising one or more antioxidant compounds; however, an antioxidant component may consist of a single antioxidant compound. In the context of the compositions of this invention, antioxidants preferably mitigate the oxidation of groups within the fusion protein that may be susceptible to oxidation.

[0120] "Chlorinating agent" is a term in the art that refers to a compound that can complex with various groups, molecules, atoms or ions, preferably in a multidentate manner, and can itself exert an antioxidant effect.

[0121] In this document, when a composition is described as “characterized by the absence of [specific component]”, it means that the composition in question is substantially free of or completely free of the said component.

[0122] The term "substantially free of" when used for a given component of a composition (e.g., "a liquid pharmaceutical composition substantially free of amino acid components") means a composition in which said component is substantially not added. As stated above, these references are independent of the presence of amino acid residues in the protein structure. When a composition is "substantially free of" a given component, the composition preferably contains no more than 0.1% by weight, preferably no more than 0.01% by weight, preferably no more than 0.001% by weight, preferably no more than 0.0001% by weight, preferably no more than 0.00001% by weight, preferably no more than 0.000001% by weight, and most preferably no more than 0.0001 parts per billion (by weight).

[0123] The term "completely free of" when used for a given component of a composition (e.g., "a liquid pharmaceutical composition completely free of amino acid components") means a composition in which none of said components are present. As stated above, such references to amino acids present in a pharmaceutical composition are unrelated to the presence of amino acid residues in the protein structure.

[0124] Preferably, unless otherwise stated, when referring to parameters that may depend on pressure and / or temperature (e.g., pH, pK) a When referring to the material state (e.g., liquid, gas, etc.), preferably, unless otherwise specified, such references refer to the parameters at standard ambient temperature and pressure (SATP). The SATP temperature is 298.15 K (25 °C, 77 °F), and the absolute pressure is 100 kPa (14.504 psi, 0.987 atm).

[0125] In this document, the specific amounts of a given component of a composition, particularly buffers or buffer systems, surfactants, sugar components, amino acid components, tension modifiers, antioxidants, and / or chelating agents, preferably refer to the amount of the relevant component in its pure anhydrous form (or the composition formed by using the said amount in its pure anhydrous form), even if such a component can be used in a non-anhydrous form when forming the composition. The amount of any corresponding non-anhydrous form (e.g., monohydrate, dihydrate, etc.) can be readily calculated by simply using a suitable multiplier. For example, unless otherwise stated (in the example, when the amount refers to trehalose dihydrate), the specified amount of trehalose refers to the anhydrous form of trehalose (or the composition formed by using a specified amount / concentration of anhydrous trehalose) with a molecular weight of 342.296 g / mol. Therefore, to calculate the corresponding amount of trehalose dihydrate required to form the same composition (with less water added), the specified amount needs to be multiplied by 378.33 / 342.296, since 378.33 is the molecular weight of trehalose dihydrate. Those skilled in the art will readily understand how to reasonably adjust the amount of diluent / water according to the form of the components used to obtain the target concentration. Clearly, this problem does not apply when specific molar amounts are specified.

[0126] In this document, the term "pharmaceutical composition" means a formulation suitable for the treatment of mammals, for example, in the sense that it does not contain any excessively toxic ingredients. In this document, reference to "composition" generally means a pharmaceutical composition as defined herein.

[0127] In this document, the term "stable" generally refers to the physical and / or chemical and / or biological stability of a component (typically an active substance or a combination thereof) during storage / preservation. For aqueous compositions of biological products, storage stability preferably means storage at 2–8°C for at least 6 months, preferably at least 12 months, and preferably up to 24 months. However, accelerated stability studies can be used to provide relevant stability information.

[0128] It should be understood that references to “treatment” or “manipulation” include prevention and relief of existing symptoms. “Manipulation” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the occurrence of the state, disorder or condition in a person who may have or is susceptible to the state, disorder or condition but has not yet experienced or displayed clinical or subclinical symptoms of the state, disorder or condition; (2) suppressing the state, disorder or condition, i.e., preventing, reducing or delaying the occurrence, development or recurrence of the disease (in respect of maintenance treatment) or at least one clinical or subclinical symptom of the disease; or (3) resolving or alleviating the disease, i.e. causing the state, disorder or condition or at least one clinical or subclinical symptom of the disease to subside.

[0129] In the context of this invention, a "therapeutic effective amount" or "effective amount" of a pharmaceutical composition refers to an amount that is effective in both prevention and treatment when administered to a mammal for the treatment of a disease or condition, and the pharmaceutical composition is effective in treating the relevant disease. The "therapeutic effective amount" will vary depending on the fusion protein of the mammal to be treated, the disease and its severity, as well as age, weight, etc.

[0130] In this document, the quantities of components and ingredients specified, whether in parts, ppm (parts per million), percentages (%, e.g., weight %) or ratios, are intended to be expressed by weight, unless otherwise stated.

[0131] When the amount or concentration of a particular component of a given composition is specified as a weight percentage (wt% or %w / w), the weight percentage refers to the weight percentage of that component relative to the total weight of the composition as a whole. Those skilled in the art will understand that the sum of the weight percentages of all components of the composition (whether specified or not) will total 100 wt%. However, in cases where not all components are listed (e.g., where the composition is referred to as “comprising” one or more specific components), the weight percentage balance may optionally be constituted by unspecified ingredients (e.g., diluents, such as water, or other non-essential but suitable additives).

[0132] In this document, the amount or concentration of a particular component may be provided as a weight / volume percentage, for example, expressed as a number between 0 and 100, followed by “%w / v”, “%(w / v)”, “w / v%”, “wt / vol%”, or “%wt / vol”, especially when the component is present in a liquid composition (suitably an aqueous solution). Those skilled in the art will readily understand that a given %w / v can be converted to other weight / volume units, such as “mg / mL”. When a component is stated as being present at 1% (w / v), the component is present at a concentration of 10 mg / mL (i.e., the mg / mL figure is 10 times the figure given by %w / v). Those skilled in the art will also understand that %w / v (especially %w / v ranges) can be restated to wt% (i.e., %w / w) (especially wt% ranges), where the density of the overall composition is approximately 1 g / cm³. 3 This is suitable for the pharmaceutical compositions of the present invention. Therefore, the weight ratio between the components can be conceived from the information provided in this application.

[0133] When a composition is said to contain multiple specified ingredients (optionally in specified concentrations), the composition may optionally include other ingredients besides those specified. However, in some embodiments, a composition that is claimed to contain multiple specified ingredients may actually consist substantially of or consist of all of the specified ingredients (optionally in specified amounts). In either case, an individual component itself may include, consist substantially of, or consist of a subcomponent or one or more subcomponents. Here, whenever the term "comprising / including" is used, it may be replaced with "consistently of" or "consisting of" as appropriate to the context.

[0134] In this document, when a composition is referred to as "consistently composed of" a particular component or multiple components, the composition preferably contains at least 70% by weight of the component, preferably at least 90% by weight, more preferably at least 95% by weight, and most preferably at least 99% by weight. Preferably, a composition referred to as "consistently composed of" a particular component consists of the component except for one or more trace impurities.

[0135] The term “about” is used to modify a parameter with a numerical definition (e.g., pH) to indicate that the parameter can vary, for example, within the experimental precision range used to determine the parameter or ± up to 5% of the specified value of the parameter, preferably ±2% of the specified value of the parameter. In a preferred embodiment, the parameter described by the term “about” corresponds to the numerical value.

[0136] In this document, unless incompatible in a given context, whenever a component capable of ionization (e.g., protonation or deprotonation) is specified, the definition of said component preferably includes any suitable salt thereof, preferably a pharmaceutically acceptable salt thereof. For example, this applies to any reference herein to buffers (e.g., citric acid or citrate), amino acids, etc. Similarly, unless incompatible in a given context, whenever a component capable of neutralization is specified, the definition of said component preferably includes its neutralized form—e.g., citric acid instead of citrate.

[0137] The "isoelectric point" (pI) represents the pH at which a given molecule (or a portion thereof) is electrically neutral from a statistical point of view—that is, it carries no net charge. pI is particularly relevant to proteins, including the fusion proteins of this invention, because the functional groups they contain can be positive, negative, neutral, or polar, depending on the dominant pH of the local environment. The pI of any given molecule or a portion thereof can be determined experimentally using methods well known in the art. However, pI can also be calculated using various methods known in the art. Preferably, pI is determined experimentally.

[0138] General points and advantages of the present invention

[0139] When developing viable antibody formulations, especially antibody fusion protein formulations, it is generally acknowledged that various factors affect the stability of the formulation. In the current context, antibody-receptor fusion further complicates formulation development because both parts of the molecule must be satisfied.

[0140] This invention arose from research work involving skillful and intuitive targeting and diligent exploration of the typically barren formulation space to reveal which key excipients should or should not be present, and their relative amounts, in order to optimally complement the relevant fusion proteins for processing and / or storage. Without such a targeted and diligent approach, formulation researchers are unlikely to design viable formulations.

[0141] The many advantages of this invention, as well as the challenges actually involved in its conception and development, will be self-evident. The inventive effort set forth in this disclosure represents a significant contribution to the art, and given that despite the aforementioned unpredictability, the invention establishes a feasible and reliable formulation space based on the embodiments and data provided herein, the contribution of this invention is proportionate to its scope of protection.

[0142] Pharmaceutical Composition

[0143] This invention provides a pharmaceutical composition. Preferably, the pharmaceutical composition is a liquid pharmaceutical composition, more preferably an aqueous pharmaceutical composition (which therefore contains water, preferably water for injection, as a diluent). However, the pharmaceutical composition may also be a lyophilized composition (i.e., and therefore preferably a solid lyophilized pharmaceutical composition). Such a lyophilized composition can preferably be reconstituted to provide a liquid pharmaceutical composition, more preferably an aqueous pharmaceutical composition. The definitions and amounts given herein may refer to one or both of liquid and / or lyophilized compositions. When the amount is specified in terms of concentration in the liquid composition (e.g., whether as a weight percentage, weight per volume, or molarity or molality), it can also be converted to a concentration ratio (whether by weight or molar ratio) in the solid composition by simple calculations known in the art, for example, by utilizing the prediction of the molecular weight of the relevant components to achieve the conversion between the two common units (e.g., parts by weight or %, or moles).

[0144] The pharmaceutical composition comprises an IgG:TGFβR fusion protein and optionally one or more pharmaceutically acceptable excipients and / or carriers. Preferably, the pharmaceutical composition comprises one or more of the following: a buffer system, a surfactant, a glycoside, an amino acid component, a tonic modulator, an antioxidant, and a chelating agent. Liquid pharmaceutical compositions (whether formulated as is or reconstituted from lyophilized formulations) preferably contain a diluent, such as water (e.g., water for injection). Preferably, the pharmaceutical composition is characterized by a pH of 4-8. Preferably, the pharmaceutical composition is characterized by a weight osmolality of 240-640 mOsm / kg, especially when the pharmaceutical composition is intended for subcutaneous injection without prior dilution.

[0145] It should be understood that compounds in a pharmaceutical composition can perform more than one function. For example, histidine, an amino acid component, can also serve as part of a buffer system. Therefore, when referring to a pharmaceutical composition comprising a buffer system and an amino acid component, such embodiments cover pharmaceutical compositions having a histidine buffer system and no other amino acid components. Thus, unless incompatible in a given context, such as as an embodiment of non-overlapping concentration ranges for specifying corresponding characteristics of a pharmaceutical composition, the listed characteristics of a pharmaceutical composition can be satisfied by one or more compounds satisfying more than one listed characteristic (i.e., multifunctional compounds). In some embodiments, the listed characteristics of a pharmaceutical composition are achieved by separate compounds. In some embodiments, the listed characteristics of a pharmaceutical composition are satisfied by one or more compounds satisfying more than one listed characteristic.

[0146] In certain embodiments, the pharmaceutical composition comprises (or consists of, optionally together with water for injection in the case of an aqueous composition) and / or is characterized by one or more of the following: IgG:TGFβR fusion protein; a buffer system; pH 4-8; a surfactant; a sugar component; an amino acid component; a tension modifier; an antioxidant; and / or a chelating agent.

[0147] Paragraphs A1 to A9 below disclose specific embodiments of the present invention.

[0148] A1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein.

[0149] The pharmaceutical composition of A2.A1 further comprises a tension modifier.

[0150] Pharmaceutical compositions of A3, A1 or A2, further characterized by a pH of 4-8.

[0151] A4. The pharmaceutical composition as described in any one of A1-A3, further comprising a buffer system.

[0152] A5. The pharmaceutical composition as described in any one of A1-A4, further comprising a surfactant.

[0153] A6. The pharmaceutical composition as described in any one of A1-A5, further comprising a sugar component.

[0154] A7. The pharmaceutical composition as described in any one of A1-A6, further comprising an antioxidant.

[0155] A8. The pharmaceutical composition as described in any one of A1-A7, further comprising an amino acid component.

[0156] A9. The pharmaceutical composition as described in any one of A1-A8, further comprising a chelating agent.

[0157] Fusion protein

[0158] The pharmaceutical composition contains IgG:TGFβR fusion protein.

[0159] The IgG:TGFβR fusion protein is preferably an IgG:TGFβR fusion protein comprising IgG fused with a soluble extracellular domain of TGFβR, more preferably a soluble extracellular domain of TGFβR2, or a fragment thereof capable of binding TGF-β.

[0160] The IgG:TGFβR fusion protein is preferably an IgG:TGFβR fusion protein, wherein the IgG has a pI of 8-10, preferably 8.5-9.5, and the TGFβR has a pI of 4.5-6, preferably 4.6-5.4.

[0161] The IgG in the IgG:TGFβR fusion protein is preferably selected from anti-PD-L1 (IgG) and anti-PD-1 (IgG), more preferably anti-PD-L1 (IgG). In some embodiments, the anti-PD-L1 (IgG) is selected from the group consisting of: (1) anti-PD-L1 (IgG) comprising three heavy chain CDRs and three light chain CDRs, wherein the three heavy chain CDRs have amino acid sequences SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3), and the three light chain CDRs have amino acid sequences SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3); (2) anti-PD-L1 (IgG) comprising three heavy chain CDRs and three light chain CDRs, wherein the three heavy chain CDRs have amino acid sequences SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:24 (CDR3); NO:3 (CDR3), wherein the three light chain CDRs have amino acid sequences SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2) and SEQ ID NO:6 (CDR3), and (3) anti-PD-L1 (IgG), which comprises three heavy chain CDRs and three light chain CDRs, wherein the three heavy chain CDRs have amino acid sequences SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2) and SEQ ID NO:29 (CDR3), and the three light chain CDRs have amino acid sequences SEQ ID NO:30 (CDR1), SEQ ID NO:31 (CDR2) and SEQ ID NO:32 (CDR3). In a preferred embodiment, anti-PD-L1 (IgG) comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDRs have amino acid sequences SEQ ID NO:1, 2 and 3, and the light chain CDRs have amino acid sequences SEQ ID NO:4, 5 and 6. In some embodiments, the light chain variable region and heavy chain variable region of the anti-PD-L1 (IgG) comprise SEQ ID NO:25 and SEQ ID NO:26, respectively. In some embodiments, the light chain sequence and heavy chain sequence of the anti-PD-L1 (IgG) correspond to (1) SEQ ID NO:7 and SEQ ID NO:16, (2) SEQ ID NO:15 and SEQ ID NO:14, or (3) SEQ ID NO:33 and SEQ ID NO:35, respectively. The IgG class of the IgG:TGFβR fusion protein is preferably selected from the group consisting of IgG1, IgG2, and IgG4, more preferably IgG1 or IgG4, and most preferably IgG1.

[0162] Preferably, the C of the IgG:TGFβR fusion protein H 3. Structural domains and bintrafusp alfa's C H The amino acid sequence of the 3-domain has greater than or equal to 85% sequence identity, greater than or equal to 90% sequence identity, greater than or equal to 95% sequence identity, or at least 96% sequence identity. Preferably, the C3 domain of the IgG:TGFβR fusion protein... H The amino acid sequence of the 3-domain is similar to that of bintrafusp alfa (C). H The 3 domains have differences of no more than 10, no more than 5, or no more than 4 amino acid residues.

[0163] Preferably, the C of the IgG:TGFβR fusion protein H 1. C of structural domains and bintrafusp alfa H The amino acid sequence of the 1 domain has greater than or equal to 80% sequence identity, greater than or equal to 85% sequence identity, greater than or equal to 90% sequence identity, or at least 91% sequence identity. Preferably, the C domain of the IgG:TGFβR fusion protein... H 1. C of structural domains and bintrafusp alfa H The 1 domain has a difference of no more than 20, no more than 10, or no more than 7 amino acid residues.

[0164] Preferably, the C of the IgG:TGFβR fusion protein H 2. Structural domains and bintrafusp alfa's C H The amino acid sequence of the 2-domain has greater than or equal to 80% sequence identity, greater than or equal to 85% sequence identity, greater than or equal to 90% sequence identity, or at least 91% sequence identity. Preferably, the C-domain of the IgG:TGFβR fusion protein... H 2. Structural domains and bintrafusp alfa's C H The two domains have a difference of no more than 20, no more than 10, or no more than 8 amino acid residues.

[0165] The variability of variable structural domains (light and heavy chains) and the entire light chain is tolerable.

[0166] The TGFβR in the IgG:TGFβR fusion protein is preferably TGFβR1 or TGFβR2, more preferably TGFβR2. In a preferred embodiment, it is an IgG:TGFβR2 fusion protein, wherein the pI of IgG is 8.5-9.5, and the pI of TGFβR2 is 4.6-5.4. In another preferred embodiment, it is an anti-PD-L1(IgG):TGFβR2 fusion protein, such as anti-PD-L1(IgG1):TGFβR2 or anti-PD-L1(IgG4):TGFβR2. Most preferably, it is anti-PD-L1(IgG1):TGFβR2. Preferably, TGFβR2 is the soluble extracellular domain of TGFβR2 or a fragment thereof capable of binding TGF-β. Preferably, TGFβR2 lacks the cytoplasmic domain of TGFβR2. In some embodiments, TGFβR2 corresponds to the wild-type human TGFβ receptor type 2 isotype A sequence (e.g., the amino acid sequence of NCBI Reference Sequence (RefSeq) accession number NP_001020018 (SEQ ID NO:9)) or the wild-type human TGFβ receptor type 2 isotype B sequence (e.g., the amino acid sequence of NCBI RefSeq accession number NP_003233 (SEQ ID NO:10)). Preferably, TGFβR2 comprises or consists of the sequence corresponding to SEQ ID NO:11 or a fragment thereof capable of binding TGFβ. For example, TGFβR2 may correspond to the full-length sequence of SEQ ID NO:11. Alternatively, it may have an N-terminal deletion. For example, N-terminal amino acids 1-26 of SEQ ID NO:11, such as 14-21 or 14-26 of the N-terminal amino acids, may be deleted. In some embodiments, N-terminal amino acids 14, 19, or 21 of SEQ ID NO:11 are deleted. Preferably, TGFβR2 comprises or consists of sequences selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. Preferably, TGFβR2 has at least 80%, at least 90%, or at least 95% sequence identity with the full-length amino acid sequence of any one of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In another preferred embodiment, TGFβR2 has at least 80% sequence identity with the full-length amino acid sequence of SEQ ID NO:11. In a preferred embodiment, the amino acid sequence of TGFβR2 differs from that of SEQ ID NO:11 by no more than 25 amino acids.

[0167] Preferably, the TGFβR of the IgG:TGFβR fusion protein has an amino acid sequence identity of 92% or more, 95% or more, 99% or more, or 100% with respect to the TGFβR of bintrafusp alfa. Preferably, the TGFβR of the IgG:TGFβR fusion protein differs from the TGFβR of bintrafusp alfa by no more than 50, 40, or 25 amino acid residues. The TGFβR of the IgG:TGFβR fusion protein preferably has 100-160 amino acid residues, more preferably 110-140 amino acid residues. In some embodiments, the amino acid sequence of TGFβR is selected from the group consisting of: the sequence corresponding to positions 1-136 of the TGFβR of bintrafusp alfa, the sequence corresponding to positions 20-136 of the TGFβR of bintrafusp alfa, and the sequence corresponding to positions 22-136 of the TGFβR of bintrafusp alfa.

[0168] Preferably, the amino acid sequence of the TGFβR in the IgG:TGFβR fusion protein has greater than or equal to 98% sequence identity with the TGFβR of bintrafusp alfa, and the C of the IgG:TGFβR fusion protein is... H 3. Structural domains and the C of bintrafuspalfa H The amino acid sequence of the 3-domain has greater than or equal to 92% sequence identity. Preferably, the amino acid sequence of TGFβR in the IgG:TGFβR fusion protein differs from that of the TGFβR in bintrafusp alfa by no more than 25 amino acid residues, and the C of the IgG:TGFβR fusion protein... H 3. Structural domains and bintrafusp alfa's C H The amino acid sequence of the 3-domain has a difference of no more than 4 amino acid residues.

[0169] Preferably, the IgG:TGFβR fusion protein includes a linker between IgG and TGFβR, which preferably contains 5-50 amino acid residues, 10-30 amino acid residues, or 20-27 amino acid residues. Preferably, this linker contains at most two different types of amino acid residues. Preferably, the linker contains glycine amino acid residues and / or serine amino acid residues. Preferably, this linker is composed of the formula (Gly... x Ser) y Gly is defined as follows: x is an integer from 1 to 6, and y is an integer from 2 to 7. Preferably, x is 4. Preferably, y is 4 or 5. Preferably, the connector is defined by formula (Gly... x Ser) yGly is defined as follows, where x is 4 and y is 4 or 5.

[0170] The IgG:TGFβR fusion protein is preferably an IgG:TGFβR2 fusion protein, which contains TGFβR2 fused at its N-terminus to the C-terminus of an IgG antibody, optionally via a linker.

[0171] Preferably, the IgG:TGFβR fusion protein is one of the IgG:TGFβR fusion proteins disclosed in WO2015 / 118175 or WO2018 / 205985. For example, the IgG:TGFβR fusion protein may comprise the light chain and heavy chain of SEQ ID NO:1 and SEQ ID NO:3 of WO2015 / 118175, respectively. In another embodiment, the IgG:TGFβR fusion protein is one of the constructs listed in Table 2 of WO2018 / 205985, such as construct 9 or 15.

[0172] Preferably, the light chain sequence and heavy chain sequence of the IgG:TGFβR fusion protein correspond to (1) SEQ ID NO:7 and SEQ ID NO:8, (2) SEQ ID NO:15 and SEQ ID NO:17, (3) SEQ ID NO:15 and SEQ ID NO:18, or (4) SEQ ID NO:33 and SEQ ID NO:34, respectively. Preferably, the amino acid sequence of the IgG:TGFβR fusion protein is identical to the amino acid sequence of bintrafusp alfa. Most preferably, the IgG:TGFβR fusion protein is bintrafusp alfa.

[0173] In one specific embodiment, the IgG:TGFβR fusion protein is characterized by:

[0174] ●TGFβR, whose amino acid sequence is greater than or equal to 95% similar to that of TGFβR in bintrafusp alfa.

[0175] Sequence identity;

[0176] ●C H 3 structural domains, which are related to the C of bintrafusp alfa. H The amino acid sequences of the three domains have greater than or equal to 92% sequence identity;

[0177] ●C H 1. Structural domain, which is related to the C of bintrafusp alfa. H The amino acid sequences of the 1-domain have greater than or equal to 90% sequence identity; and

[0178] ●C H2. Structural domains, which are related to the C of bintrafusp alfa. H The amino acid sequence of the 2-domain has greater than or equal to 90% sequence identity.

[0179] In one specific embodiment, the IgG:TGFβR fusion protein is characterized by:

[0180] ●TGFβR, which differs from the TGFβR of bintrafusp alfa by no more than 25 amino acid residues;

[0181] ●C H 3 structural domains, which are related to the C of bintrafusp alfa. H The amino acid sequence of the 3-domain structure has a difference of no more than 4 amino acid residues;

[0182] ●C H 1. Structural domain, which is related to the C of bintrafusp alfa. H The amino acid sequence of domain 1 differs by no more than 7 amino acid residues; and

[0183] ●C H 2. Structural domains, which are related to the C of bintrafusp alfa. H The amino acid sequence of the 2-domain has a difference of no more than 8 amino acid residues.

[0184] For region-specific comparisons, such as C for bintrafusp alfa H 1. C H 2 and / or C H The amino acid sequence of the 3-domain structure (and optionally the TGFβR domain) can be used. Figure 1A or Figure 1B The specific sequence shown is assigned to a specific region. In some implementations, for the purpose of region-specific comparisons, such as with respect to C in bintrafusp alfa... H 1. C H 2 and / or C H The amino acid sequence of the 3-domain structure (and optionally the TGFβR domain) can be used. Figure 1B The specific sequence shown is assigned to a specific region.

[0185] For bintrafusp alfa's C H 2. Region-specific comparison of the amino acid sequences of the structural domains can be used. Figure 1A or Figure 1B The assignment shown to C H2. Sequence of structural domains. In some implementations, for bintrafusp alfa, C H 2. Region-specific comparison of the amino acid sequences of the structural domains can be used. Figure 1B The assignment shown to C H 2. Sequence of structural domains.

[0186] although Figure 1A and Figure 1B The text illustrates the case of assigning specific sequences to specific regions, but region-specific comparisons are possible, such as those concerning bintrafusp alfa (C). H 1. C H 2 and / or C H The amino acid sequence of the 3-domain (and optionally the TGFβR domain), without reference to any of the figures in which the antibody sequence is routinely assigned based on these regions.

[0187] The pharmaceutical composition preferably contains IgG:TGFβR fusion protein (e.g., IgG:TGFβR or anti-PD-L1(IgG):TGFβR2) in amounts of 1-200 mg / mL, 5-150 mg / mL, 7-70 mg / mL, 5-15 mg / mL, 15-65 mg / mL, 15-30 mg / mL, 35-65 mg / mL, 35-45 mg / mL, 45-55 mg / mL, 55-65 mg / mL, 40-120 mg / mL, 75-115 mg / mL, or 95-105 mg / mL. In some embodiments, the pharmaceutical composition contains about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, or about 100 mg / mL of anti-PD-L1(IgG):TGFβR2 fusion protein.

[0188] diluent

[0189] The pharmaceutical composition preferably contains a diluent. The composition may contain one or more pharmaceutically acceptable diluents, or mixtures thereof. However, most preferably, the composition is an aqueous composition. Most preferably, the diluent is water, and preferably water alone. The water is preferably water for injection (WFI).

[0190] Preferably, the diluent can constitute a balance of the components in any composition, for example, such that the total weight percentage of all components is 100%. Preferably, any concentration given herein in relation to any component of the composition represents the concentration of said component in the diluent mixed (and preferably dissolved) with any other component.

[0191] The compositions of the present invention are preferably solutions and preferably (substantially or completely) free of particles or precipitates.

[0192] However, in one embodiment, the pharmaceutical composition is anhydrous or contains at most 10% by weight of water, preferably at most 5% by weight, more preferably at most 2% by weight, and more preferably at most 1% by weight. Such embodiments can be solid pharmaceutical compositions or lyophilized pharmaceutical compositions, for example, which can be reconstituted (preferably by adding water and / or other relevant diluents, such as intravenous infusions or saline solutions) before use, administration, or (preferably short-term) storage. Such lyophilized formulations can be reconstituted to provide the aqueous pharmaceutical compositions disclosed herein (e.g., having components present or absent at concentrations specified herein).

[0193] Buffer system

[0194] The pharmaceutical composition preferably comprises a buffer system. When a small amount of (strong) acid or (strong) base is added to (or generated therein, possibly as a result of degradation of one or more components of the pharmaceutical composition (most likely its fusion protein), the buffer system preferably acts as a pH buffer inert to pH changes. Therefore, the buffer system preferably maintains a substantially constant pH of the composition over time, thereby preferably mitigating pH-triggered degradation pathways. Preferably, the pharmaceutical composition has sufficient buffering capacity to resist pH changes of ≥1 pH unit during storage at 2-8°C for 6 months, preferably ≥0.5 pH units, and most preferably ≥0.2 pH units (under equivalent storage conditions).

[0195] Preferably, the composition is a buffer solution whose pH is stabilized by a buffering agent, which is a weak acid or weak base, and a conjugate acid or conjugate base of the buffering agent, depending on whether the buffering agent itself is a base or an acid. In general, a buffering agent and its acid / base conjugate can be considered a “buffer system,” although in some embodiments where more than one buffer system exists, the composition may contain a variety of different buffering agents and corresponding acid / base conjugates. Therefore, the composition preferably comprises a “buffer system” (preferably comprising a buffering agent and its acid / base conjugate), and any concentration specified regarding the buffer system generally refers to the combined concentration of one or more buffering agents and any one or more acid / base conjugates.

[0196] The pharmaceutical composition preferably comprises a buffer system, which includes one or more buffer systems. For example, the buffer system may be a dual buffer system (e.g., histidine-acetate, phosphate-citrate). Most preferably, the pharmaceutical composition comprises a buffer system containing only one buffer system. However, the pharmaceutical composition may be characterized by the absence of a buffer system (or any one or more of those specifically described herein with reference to buffer systems). For example, the pharmaceutical composition may be sufficiently stable in the absence of a buffer system, wherein the fusion protein provides sufficient self-buffering, which preferably occurs at higher concentrations of the fusion protein.

[0197] In some embodiments, the buffer system is selected from single-proton buffer systems (e.g., acetate buffer), multi-proton buffer systems (e.g., phosphate buffer), and amphoteric buffer systems (e.g., amino acid buffers, such as histidine), inorganic buffer systems (e.g., ammonium buffer, bicarbonate buffer, carbonate buffer, borate buffer, phosphate buffer), organic buffer systems (e.g., carboxylate buffer, organic ammonium buffer, alkanol ammonium buffer, amphoteric buffer, amino acid buffer, aromatic nitrogen buffer, sugar buffer), and any combination thereof.

[0198] In other embodiments, the buffer system is selected from monocarboxylate buffer systems (e.g., acetate buffer, formate buffer, lactate buffer, salicylate buffer, benzoate buffer), dicarboxylate buffer systems (e.g., succinate buffer, maleate buffer), malate buffer, fumarate buffer, tartrate buffer, adipic acid buffer, hexanedioate buffer), tricarboxylate buffer systems (e.g., citrate buffer), zwitterionic buffer systems (e.g., amino acid buffer, zwitterionic sulfonate buffer, such as N-(2-acetamido)-2- Aminotaurine sulfonic acid (ACES) buffer, 2-aminotaurine sulfonic acid (AES) buffer, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, N,N-bis-(2-hydroxyethyl)-2-aminotaurine sulfonic acid (BES) buffer, 3-(cyclohexylamino)-propanesulfonic acid (CAPS) buffer, amino acid buffer systems (e.g., histidine buffer, glycine buffer, lysine buffer, glycylglycine buffer, N-[Tris(hydroxymethyl)-methyl]-glycine (tricine) buffer, glutamate buffer, aspartate buffer, etc. -Bis(2-hydroxyethyl)-glycine (Bicine) buffer, N-(2-acetamido)-iminodiacetic acid (ADA) buffer, aromatic nitrogen buffer systems (e.g., imidazole, pyridine buffers) and alkanol ammonium buffer systems (e.g., aminomethylpropanol (AMP) buffer, aminomethylpropanediol (AMPD) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris) buffer, 1,3-bis[tris(hydroxymethyl)-methylamino]propane (bistripropane) buffer).

[0199] Suitably, the buffer system is a buffer system selected from the group consisting of: histidine buffer, phosphate buffer, succinate buffer, citrate buffer, acetate buffer, gluconate buffer, Tris buffer, aspartate buffer, glutamate buffer, tartrate buffer, malate buffer, maleate buffer, fumarate buffer, histidine-acetate buffer, phosphate-citrate buffer, and any combination thereof. Preferably, the buffer system is a buffer system selected from the group consisting of: histidine buffer, phosphate buffer, succinate buffer, citrate buffer, and any combination thereof. More preferably, the buffer system is a buffer system comprising a single buffer selected from the group consisting of: histidine buffer, phosphate buffer, succinate buffer, and citrate buffer.

[0200] The pharmaceutical composition may comprise a 1-100 mM buffer system, a 2-70 mM buffer system, a 3-50 mM buffer system, a 4-30 mM buffer system, a 5-20 mM buffer system, a 6-14 mM buffer system, or preferably, a 10 mM buffer system.

[0201] The pharmaceutical composition may contain a buffer system in the following molar ratios to the fusion protein: 1280:1 to 3:1, 370:1 to 9:1, 260:1 to 11:1, 100:1 to 15:1, or preferably 70:1 to 20:1.

[0202] Most preferably, the buffer system is or contains a histidine buffer system. For example, the buffer system is or contains a 5-60 mM histidine buffer system, a 5-15 mM histidine buffer system, or preferably about 10 mM histidine buffer system. In other embodiments, the composition contains a histidine buffer system in a molar ratio of 1280:1 to 3:1 or 370:1 to 9:1 to the fusion protein.

[0203] Preferably, the buffer system is or comprises a phosphate buffer system. For example, the buffer system is or comprises a 5-60 mM phosphate buffer system or a 5-15 mM phosphate buffer system. In other embodiments, the composition comprises a phosphate buffer system to the fusion protein in a molar ratio of 1280:1 to 3:1 or 370:1 to 9:1.

[0204] Preferably, the buffer system is or includes a succinate buffer system. For example, the buffer system is or includes a 5-60 mM succinate buffer system or a 5-15 mM succinate buffer system. In other embodiments, the composition includes a succinate buffer system in a molar ratio of 1280:1 to 3:1 or 370:1 to 9:1 to the fusion protein.

[0205] Preferably, the buffer system is or includes a citrate buffer system. For example, the buffer system is or includes a 5-60 mM citrate buffer system or a 5-15 mM citrate buffer system. In other embodiments, the composition contains a citrate buffer system in a molar ratio of 1280:1 to 3:1 or 370:1 to 9:1 to the fusion protein.

[0206] Preferably, the buffer system does not contain acetate buffer.

[0207] The quantities and concentrations associated with a buffer system refer to the total amount and concentration of all buffer systems, unless only a single buffer system is specified. The concentration of the stated or any particular buffer system can be replaced by a molar ratio.

[0208] pH

[0209] The pharmaceutical composition preferably has a pH of 4-9. Although the fusion protein and any excipients in the pharmaceutical composition can be tolerated and remain fairly stable within a certain pH range, certain pH values ​​are particularly advantageous, especially in the presence of specific excipients or combinations thereof.

[0210] In some embodiments, the pharmaceutical composition has a pH of 4-8, 4.8-7.8, 5-7, 4.9-6.8, 4.5-6.0, 4.8-6.5, 5-5.4, 5.2-6.2, 5.3-6.3, 5.2-5.8, 5.6-5.8, 5.9-6.1, 5.2-6.2, or 5.4-6.0, or preferably, a pH of 5.4-5.6 or 5.8-6.0. In some embodiments, the pharmaceutical composition has a pH of about 5.5 or about 5.9.

[0211] One of the challenges encountered in the formulation of IgG:TGFβR fusion protein compositions is the significant difference in pI between the IgG moiety (typically around 8-10, 8.5-9.5, or around 9.1) and the TGFβR moiety (typically around 4.5-6, 4.6-5.4, or around 4.9). It is generally desirable to formulate biologics at a pH greater than 1-2 units above the pI of the biologic-related moiety. Optimizing the pH of bifunctional biologics, where the pI of their functional moieties shows a significant difference, is particularly difficult. In the case of IgG:TGFβR fusion proteins, the TGFβR moiety has a significant influence on formulation considerations and may be less stable than the IgG moiety. In particular, the IgG moiety of bintrafusp alfa has been found to be quite tolerant of stress conditions and to be long-term stable even in liquid / non-lyophilized forms. In contrast, when the IgG moiety of bintrafusp alfa is fused with the TGFβR moiety, the molecule is found to precipitate more readily, and particle formation and phase separation are observed in the formulation. This suggests that the TGFβR moiety is less stable than the IgG moiety, or at least acts as an unstable component together with it. This reinforces the idea that the pH of IgG:TGFβR fusion protein compositions should have a pH particularly far removed from the pI of the TGFβR moiety, for example, a pH outside of 3.9–5.9. However, surprisingly, pharmaceutical compositions with a pH close to the pI of the TGFβR moiety of the IgG:TGFβR fusion protein were found to be highly stable.

[0212] Therefore, the pharmaceutical composition preferably has a pH within 2 pH units of the pI of the TGFβR moiety of the fusion protein, more preferably within 1 pH unit (i.e., the difference between the pH of the composition and the pI of the TGFβR moiety is less than 2 units or 1 unit, respectively). The pharmaceutical composition preferably has a pH that differs from the pI of the IgG moiety of the fusion protein by greater than or equal to 2 pH units, or more preferably greater than or equal to 3 pH units. For example, the composition may have a pH of 5.4-6.0, wherein the pI of the TGFβR moiety is 4.4-5.0 and the pI of the IgG moiety is 8.4-9.5.

[0213] Although this invention covers any buffer system with or without the use of the buffer system defined herein, as long as the pH is as defined herein, the histidine buffer system is the most preferred buffer system, with the most preferred pH being 5.2-6.2.

[0214] surfactants

[0215] The pharmaceutical composition preferably contains a surfactant. The pharmaceutical composition preferably contains at most one surfactant. In the context of this invention, surfactants can inhibit one or more degradation pathways of the fusion protein or its components, such as unfolding (and consequent aggregation), aggregation, and sometimes even fragmentation. Surfactants can promote the dissolution of the fusion protein. However, the pharmaceutical composition may be characterized by the absence of a surfactant (or any one or more of those specifically described herein with reference to surfactants).

[0216] Preferably, the surfactant is a nonionic surfactant, such as selected from the group consisting of: fatty alcohols, fatty alcohol ethers, fatty acid esters, fatty acid amides, polyoxyethylene alkyl ethers, polyoxyethylene alkyl ethers, nonionic block copolymers, α-tocopherol and any combination thereof, or selected from the group consisting of: sorbitan esters (Span), ethoxylated sorbitan esters (polysorbate) and block alkoxylates.

[0217] In some embodiments, the surfactant is an ethoxylated fatty acid ester surfactant. In other embodiments, the surfactant is a surfactant selected from the group consisting of one or more polysorbate esters, one or more poloxamers, and one or more kolliphors. Preferably, it is a kolliphor surfactant or a polysorbate surfactant. Preferably, the kolliphor is kolliphor 188, and the polysorbate is polysorbate 20 or polysorbate 80 surfactant. Most preferably, the surfactant is polysorbate 20.

[0218] The pharmaceutical composition preferably contains 0.01-2 mg / mL surfactant, 0.05-1.5 mg / mL surfactant, 0.1-0.6 mg / mL surfactant, 0.25-0.75 mg / mL surfactant, 0.4-0.6 mg / mL surfactant, 0.4-1.2 mg / mL surfactant, or 0.8-1.1 mg / mL surfactant.

[0219] The pharmaceutical composition preferably contains a surfactant, wherein the molar ratio of the surfactant to the fusion protein is 30:1 to 1:70, 12:1 to 1:3, 17:1 to 1:1, 5:1 to 1:2, or 7:1 to 1:1.

[0220] The pharmaceutical composition preferably comprises 0.01-2 mg / mL polysorbate 20, 0.05-1.5 mg / mL polysorbate 20, 0.05-0.3 mg / mL polysorbate 20, 0.05-0.15 mg / mL polysorbate 20, 0.1-0.7 mg / mL polysorbate 20, 0.3-0.7 mg / mL polysorbate 20, 0.4-0.6 mg / mL polysorbate 20, 0.4-1.3 mg / mL polysorbate 20, 0.8-1.2 mg / mL polysorbate 20, 0.9-1.1 mg / mL polysorbate 20, or most preferably about 0.5 mg / mL polysorbate 20.

[0221] The pharmaceutical composition preferably comprises polysorbate 20, wherein the molar ratio of polysorbate 20 to the fusion protein is 30:1 to 1:70, 12:1 to 1:3, 17:1 to 1:1, 5:1 to 1:2, or 7:1 to 1:1.

[0222] The amounts and concentrations associated with surfactants refer to the total amount and concentration of all surfactants, unless only a single surfactant is specified. The concentration of the stated or any specific surfactant can be replaced by a molar ratio.

[0223] Sugar components

[0224] Pharmaceutical compositions preferably contain a sugar component, such as a lyophilization protectant sugar component. In the context of this invention, sugar components may function in one or more ways in the pharmaceutical composition. For example, they may act as lyophilization protectants during the lyophilization process. For example, sugar components may provide tonicity to maintain the osmotic molar concentration within a desired range (e.g., isotonicity for undiluted injectable formulations—e.g., 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, most preferably 270-310 mOsmol / L). Using sugar components to promote tonicity may be particularly useful where high ionic strength is less desirable, although compositions of this invention generally tend to favor higher ionic strengths for higher concentrations of fusion proteins. Sugar components can promote the solubility of fusion proteins and / or other excipients in the pharmaceutical composition. Sugar components can provide stabilizing effects on fusion proteins in the pharmaceutical composition, for example, improving conformational stability (e.g., reducing protein unfolding events that can increase the likelihood of eventual aggregation), reducing aggregation, and / or reducing fragmentation. However, in some embodiments, the pharmaceutical composition is characterized by the absence of sugar components (or any one or more of those specifically described herein with respect to sugar components).

[0225] Preferably, the sugar is nonionic and / or does not contain ionizable groups.

[0226] In some embodiments, the sugar component is a non-reducing sugar component. The sugar component may also comprise one or more sugars and / or sugar alcohols. In some embodiments, the sugar component comprises one or more sugars or one or more sugar alcohols. Preferably, the sugar component consists of a single compound. In other embodiments, the sugar component comprises at most one sugar or at most one sugar alcohol.

[0227] Preferably, the sugar component is selected from the group consisting of trehalose, sucrose, mannitol and sorbitol, and more preferably from trehalose or sucrose.

[0228] In some embodiments, the sugar component is a sugar, which may be selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and complex carbohydrates. Preferably, the sugar component is a disaccharide, such as a non-reducing disaccharide. More preferably, the sugar component is a disaccharide selected from trehalose or sucrose. Preferably, the sugar component is sucrose. Preferably, the sugar component is trehalose.

[0229] In some embodiments, the sugar component is a sugar alcohol, such as a (3-12C) sugar alcohol, a (3-6C) sugar alcohol, or a (5-6C) sugar alcohol, selected from mannitol, sorbitol, arabinitol, xylitol, ribitol, and inositol. In some embodiments, the sugar component is a sugar alcohol selected from mannitol or sorbitol.

[0230] The pharmaceutical composition preferably comprises 30-400 mM of sugar component, 40-300 mM of sugar component, 40-100 mM of sugar component, 40-60 mM of sugar component, 90-290 mM of sugar component, 100-200 mM of sugar component, 130-170 mM of sugar component, 200-300 mM of sugar component, 230-270 mM of sugar component, about 159 mM of sugar component, about 100 mM of sugar component, or about 50 mM of sugar component.

[0231] The pharmaceutical composition preferably contains sugar components in the following molar ratios to the fusion protein: 7300:1 to 50:1, 3700:1 to 70:1, 1000:1 to 100:1, 1000:1 to 500:1, 500:1 to 180:1, 2900:1 to 2700:1, 800:1 to 600:1, 500:1 to 300:1, or 300:1 to 100:1.

[0232] The pharmaceutical composition preferably comprises 40-300 mM trehalose, 40-100 mM trehalose, 40-60 mM trehalose, 65-85 mM trehalose, 70-130 mM trehalose, 90-110 mM trehalose, 100-200 mM trehalose, 140-180 mM trehalose, or 150-170 mM trehalose. In some embodiments, the pharmaceutical composition comprises trehalose at concentrations of about 50 mM, about 75 mM, about 100 mM, or about 159 mM. The pharmaceutical composition preferably comprises trehalose in the following molar ratios of trehalose to the fusion protein: 7300:1 to 50:1, 3700:1 to 70:1, 1000:1 to 100:1, 1000:1 to 500:1, or 500:1 to 180:1. Most preferably, the pharmaceutical composition contains trehalose as the sole sugar component, with a preferred concentration of 40-200 mM.

[0233] The pharmaceutical composition preferably contains 50-300 mM sucrose, 150-290 mM sucrose, 220-280 mM sucrose or 240-260 mM sucrose.

[0234] The pharmaceutical composition preferably contains 40-300 mM mannitol, 40-100 mM mannitol, 40-60 mM mannitol, 80-120 mM mannitol, 100-200 mM mannitol, 210-290 mM mannitol, or 240-260 mM mannitol.

[0235] The pharmaceutical composition preferably comprises 40-300 mM sorbitol, 40-100 mM sorbitol, 40-60 mM sorbitol, 80-120 mM sorbitol, 100-200 mM sorbitol, 210-290 mM sorbitol, or 240-260 mM sorbitol.

[0236] The quantities and concentrations related to sugar components refer to the total amount and concentration of all sugar components unless only a single sugar component is specified. The concentration of the stated or any specific sugar component can be replaced by a molar ratio.

[0237] amino acid components

[0238] The pharmaceutical composition preferably comprises an amino acid component. Amino acids can be used to adjust the osmotic molar concentration of the pharmaceutical composition, for example, to bring the osmotic molar concentration within a preferred range (e.g., for isotonicity of undiluted injectable formulations—e.g., 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, most preferably 270-310 mOsmol / L). Amino acids can also promote the solubility of fusion proteins and / or other excipients in the pharmaceutical composition. Amino acids can also provide stabilizing effects on the fusion proteins of the pharmaceutical composition, for example, improving conformational stability (e.g., reducing protein unfolding events that could increase the likelihood of eventual aggregation), reducing aggregation, and / or reducing fragmentation. One or more amino acids (e.g., arginine) or any salt thereof (e.g., arginine hydrochloride) can replace some or all of the tonic modifiers (e.g., NaCl) and / or some or all of the sugar components (e.g., trehalose). Preferably, the pharmaceutical composition also comprises an amino acid component other than histidine, wherein histidine serves as a buffer system or a portion thereof, and / or wherein methionine serves as an antioxidant component or a portion thereof.

[0239] However, the pharmaceutical composition may be characterized by the absence of an amino acid component (or any one or more of those specifically referred to herein with respect to an amino acid component), preferably with histidine as an optional exception, wherein histidine is used as a buffer system or a portion thereof, or with methionine as an optional exception, wherein methionine is used as an antioxidant component or a portion thereof, preferably with histidine and methionine as optional exceptions, wherein they are used as a buffer system or a portion thereof and as an antioxidant component or a portion thereof, respectively.

[0240] Preferably, the amino acid component comprises one or more amino acids, particularly one or more amino acids different from histidine (where histidine is included), for example as a buffer system or part thereof, and / or one or more amino acids different from methionine (where methionine is included), for example as an antioxidant component or part thereof. Preferably, the amino acid component comprises at most one amino acid or at most one amino acid different from histidine (where histidine is included), for example as a buffer system or part thereof, and / or at most one amino acid different from methionine (where methionine is included), for example as an antioxidant component or part thereof. Preferably, the amino acid component comprises a single amino acid, or a single amino acid different from histidine (where histidine is included), for example as a buffer system or part thereof, and / or a single amino acid different from methionine (where methionine is included), for example as an antioxidant component or part thereof. Preferably, the amino acid component comprises L-amino acids. It is preferred that the amino acid constituting the amino acid component is an L-amino acid.

[0241] The amino acid component, or any amino acid thereof, may be a pharmaceutically acceptable salt. For example, in the case of arginine, arginine can actually be provided in the form of arginine hydrochloride.

[0242] Preferably, the amino acid component comprises amino acids selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof.

[0243] Preferably, the amino acid component includes arginine.

[0244] Preferably, the amino acid component comprises lysine.

[0245] Preferably, the amino acid component includes proline.

[0246] Preferably, the amino acid component includes glutamic acid.

[0247] The amino acid is preferably a charged amino acid (i.e., an amino acid with a net positive or negative charge). Preferably, the amino acid component comprises an amino acid salt (e.g., arginine hydrochloride).

[0248] The pharmaceutical composition preferably comprises 10-300 mM amino acid components, 20-260 mM amino acid components, 30-110 mM amino acid components, 30-60 mM amino acid components, 35-95 mM amino acid components, 40-170 mM amino acid components, 50-200 mM amino acid components, 60-140 mM amino acid components, 110-190 mM amino acid components, 140-180 mM amino acid components, or about 50 mM amino acid components.

[0249] The pharmaceutical composition preferably contains amino acid components in the following molar ratios to the fusion protein: 5500:1 to 18:1, 2000:1 to 54:1, 3500:1 to 200:1, 500:1 to 100:1, or 900:1 to 400:1.

[0250] The pharmaceutical composition preferably comprises 20-300 mM arginine, 30-60 mM arginine, 30-50 mM arginine, 40-60 mM arginine, 60-80 mM arginine, 35-95 mM arginine, 80-120 mM arginine, 100-140 mM arginine, 120-180 mM arginine, 150-170 mM arginine, 200-300 mM arginine, or preferably about 50 mM arginine or about 75 mM arginine. The pharmaceutical composition preferably comprises arginine in the following molar ratios of arginine to the fusion protein: 5500:1 to 18:1, 2000:1 to 54:1, 3500:1 to 200:1, 500:1 to 100:1, or 900:1 to 400:1.

[0251] The pharmaceutical composition preferably contains 20-200mM lysine, 30-150mM lysine, 30-80mM lysine, 40-60mM lysine, 60-90mM lysine, 70-130mM lysine, or 90-110mM lysine.

[0252] The pharmaceutical composition preferably contains 20-200 mM proline, 30-150 mM proline, 30-80 mM proline, 40-60 mM proline, 60-90 mM proline, 70-130 mM proline, or 90-110 mM proline.

[0253] The pharmaceutical composition preferably contains 20-200mM glutamic acid, 30-150mM glutamic acid, 30-80mM glutamic acid, 40-60mM glutamic acid, 60-90mM glutamic acid, 70-130mM glutamic acid, or 90-110mM glutamic acid.

[0254] The quantities and concentrations related to amino acid components refer to the total amount and concentration of all amino acid components, unless only a single amino acid component is specified. The concentration of the stated or any specific amino acid component can be replaced by a molar ratio.

[0255] Ionic strength

[0256] The pharmaceutical compositions of the present invention preferably have a non-zero ionic strength. Ionic strength is believed to promote the solubility of fusion proteins, particularly their TGFβR moiety. A certain level of ionic strength can reduce fragmentation. A certain level of ionic strength can reduce aggregation.

[0257] Ionic strength can preferably be defined as "molar ionic strength (I)", which is a function of the concentrations of all ions present in a given composition (preferably a solution, more preferably an aqueous solution). Molar ionic strength (I) can be defined by the following equation:

[0258]

[0259] Where c i It is the molar concentration (mol / L) of ion i, z i It is the charge number of ion i. The 1 / 2 multiplier reflects the fact that the summation is performed over all ions, thus including polar, anion, and cation.

[0260] For example, if NaCl is the only electrolyte contributing to the ionic strength of the composition, then the ionic strength is equal to the concentration of NaCl (i.e., 100 mM NaCl = 100 mM ionic strength), because the cations and anions of each NaCl are single-charged—therefore, for a 60 mM NaCl solution, the ionic strength is as follows:

[0261]

[0262] If the salt (e.g., MgSO4) is the only electrolyte contributing to the ionic strength of the composition, then the ionic strength is four times that of the equivalent molar concentration of sodium chloride, because both the cations and anions are doubly charged—therefore, for a 60 mM MgSO4 solution, the ionic strength is as follows:

[0263]

[0264] Therefore, multivalent ions contribute a relatively larger share of ionic strength than monovalent ions.

[0265] Amino acid salts (such as arginine hydrochloride) can also contribute to ionic strength. Buffers (or their ion-buffering substances—such as conjugate acids and / or conjugate bases) may also contribute to ionic strength.

[0266] The ionic strength of the composition is suitably equal to the sum of the ionic contributions from the tension modifier and (if present) the buffer system (as defined by the equation above). The ionic strength of the composition can be equal to any concentration of the tension modifier as defined herein plus the molar concentration of any buffer system.

[0267] In some embodiments, the compositions of the present invention may be characterized by unbuffered (or tension modulator-based) ionic strength. In this case, the unbuffered (or tension modulator-based) ionic strength of the composition is preferably equal to any concentration of the tension modulator as defined herein. Furthermore, in this case, the ratio of the unbuffered (or tension modulator-based) ionic strength to the molar concentration of the fusion protein is preferably the same as any molar ratio of the tension modulator to the fusion protein as defined herein.

[0268] The preferred ionic strength of the pharmaceutical composition is 5-250 mM, 10-200 mM, 20-170 mM, 20-80 mM, 20-60 mM, 30-50 mM, 30-130 mM, 80-150 mM, 90-100 mM, 100-170 mM, 110-130 mM, 150-170 mM, 130-170 mM, about 40 mM, about 60 mM, or about 100 mM.

[0269] The pharmaceutical composition preferably has an ionic strength in a molar ratio to the fusion protein of the following ionic strengths: 3700:1 to 15:1, 1000:1 to 70:1, 910:1 to 200:1, 910:1 to 540:1, 320:1 to 220:1, 410:1 to 320:1, 520:1 to 390:1, 700:1 to 450:1, 680:1 to 720:1, 460:1 to 420:1, or 290:1 to 250:1.

[0270] The ionic strength is preferably provided at least partially or entirely by sodium chloride. The ionic strength is preferably provided at least partially or entirely by sodium chloride and arginine (or its salt). The ionic strength is preferably provided at least partially or entirely by sodium chloride and / or amino acid salts (e.g., arginine or arginine salts).

[0271] Tension regulator

[0272] Pharmaceutical compositions preferably contain a tonic modifier, such as a non-buffered tonic modifier (i.e., a tonic modifier that does not provide pH buffering, unlike a buffer salt, which may itself contribute to overall tonicity to some extent). In the context of this invention, the tonic modifier can be used to adjust the osmotic molar concentration of the pharmaceutical composition, for example, to bring the osmotic molar concentration within a preferred range (e.g., for isotonicity of undiluted injectable formulations—e.g., 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, most preferably 270-310 mOsmol / L). Similarly, tonic modifiers, especially ionic tonic modifiers (e.g., salt tonic modifiers), can be used to provide ionic strength. The tonic modifier can also promote the solubility of fusion proteins and / or other excipients in the pharmaceutical composition. The tonic modifier can also provide stabilizing effects on fusion proteins in the pharmaceutical composition, for example, improving conformational stability (e.g., reducing protein unfolding events that can increase the likelihood of eventual aggregation), reducing aggregation, and / or reducing fragmentation.

[0273] However, in some embodiments, the pharmaceutical composition is characterized by the absence of a (non-buffered) tonic modulator (or any one or more of those specifically described herein with respect to tonic modulators). Alternatively, some tonic modulators, particularly non-buffered salt tonic modulators (e.g., NaCl), may be replaced by some amino acid components and / or some sugar components, especially where said components provide their own tonic modulating effects.

[0274] Tension regulators may be or include tension-regulating lyophilization protectants. Tension-regulating lyophilization protectants are one or more compounds that provide tension regulation of the composition and also act as lyophilization protectants, for example, during lyophilization (particularly during its drying phase). Lyophilization protection is well known in the art and may include, for example, compounds that stabilize the active ingredient and / or stabilize the lyophilized cake (e.g., prevent it from collapsing during drying) during the drying phase of lyophilization. Tension-regulating lyophilization protectants may contain or consist of sugars or sugar polyols, such as sugars or sugar polyols as defined herein as "sugar component". Tension-regulating lyophilization protectants may contain or consist of amino acids, such as "amino acid component" as defined herein. Trehalose is an example of a tension-regulating lyophilization protectant. Arginine can also be used as a lyophilization protectant. A composition containing a combination of trehalose and arginine may be advantageous.

[0275] Preferably, the tension modifier includes tension modifiers selected from the group consisting of: salt tension modifiers, metal salt tension modifiers, non-buffered salt tension modifiers, metal halide salt tension modifiers, alkali metal or alkaline earth metal halide salt tension modifiers, alkali metal halide salt tension modifiers, and alkali metal halide salt tension modifiers selected from sodium chloride or potassium chloride, preferably sodium chloride, and any combination thereof.

[0276] Preferably, the tension modifier is an ionic tension modifier and thus directly contributes to the ionic strength of the composition. Preferably, the tension modifier is a non-buffered ionic tension modifier.

[0277] The pharmaceutical composition preferably comprises 5-250 mM of a tension modifier, 10-200 mM of a tension modifier, 20-170 mM of a tension modifier, 20-80 mM of a tension modifier, 20-60 mM of a tension modifier, 50-70 mM of a tension modifier, 30-50 mM of a tension modifier, 30-130 mM of a tension modifier, 80-150 mM of a tension modifier, 90-110 mM of a tension modifier, 90-100 mM of a tension modifier, 100-170 mM of a tension modifier, 110-130 mM of a tension modifier, 150-170 mM of a tension modifier, 130-170 mM of a tension modifier, about 40 mM of a tension modifier, about 60 mM of a tension modifier, or about 100 mM of a tension modifier.

[0278] The pharmaceutical composition preferably contains a tension modifier in the following molar ratios to the fusion protein: 3700:1 to 15:1, 1000:1 to 70:1, 910:1 to 200:1, 910:1 to 540:1, 320:1 to 220:1, 410:1 to 320:1, 520:1 to 390:1, 700:1 to 450:1, 680:1 to 720:1, 460:1 to 420:1, or 290:1 to 250:1.

[0279] The pharmaceutical composition preferably comprises 5-250 mM sodium chloride, 10-200 mM sodium chloride, 20-170 mM sodium chloride, 30-90 mM sodium chloride, 20-60 mM sodium chloride, 30-50 mM sodium chloride, 50-70 mM sodium chloride, 35-45 mM sodium chloride, 30-130 mM sodium chloride, 80-150 mM sodium chloride, 90-110 mM sodium chloride, 100-200 mM sodium chloride, 110-130 mM sodium chloride, 150-170 mM sodium chloride, 130-170 mM sodium chloride, about 40 mM sodium chloride, about 60 mM sodium chloride, or about 100 mM sodium chloride.

[0280] The pharmaceutical composition preferably contains sodium chloride in the following molar ratios of sodium chloride to fusion protein: 3700:1 to 15:1, 1000:1 to 70:1, 910:1 to 200:1, 910:1 to 540:1, 600:1 to 250:1, 320:1 to 220:1, 410:1 to 320:1, 520:1 to 390:1, 700:1 to 450:1, 680:1 to 720:1, 460:1 to 420:1, or 290:1 to 250:1.

[0281] The amounts and concentrations related to tension modifiers refer to the total amount and concentration of all tension modifiers, unless only a single tension modifier is specified. The concentration of the stated or any specific tension modifier can be replaced by a molar ratio.

[0282] Typically, in the context of the formulations of this invention, the only viable component, besides a buffer system, for providing ionic strength is one or more tonicotinic agents, such as sodium chloride and / or charged amino acids (e.g., arginine, or arginine hydrochloride, or other amino acid salts). NaCl is considered a particularly advantageous component as a tonicotinic agent because it generally contributes to the solubility of fusion proteins in aqueous media, although it may also be involved in charge shielding, particularly the charge shielding of certain groups on the acceptor portion of the fusion protein. Experiments have frequently demonstrated that formulations generally benefit from higher concentrations of NaCl as the concentration of the fusion protein increases. For example, 5-15 mg / mL of fusion protein may benefit from 30-50 mM NaCl; 35-55 mg / mL of fusion protein may benefit from 50-70 mM NaCl; and 50-70 mg / mL of fusion protein may benefit from 130-170 mM NaCl.

[0283] antioxidants

[0284] The pharmaceutical composition preferably contains an antioxidant. The antioxidant can inhibit oxidative degradation pathways that open to the fusion protein and / or other components of the pharmaceutical composition. For example, the antioxidant can inhibit the oxidation of the fusion protein, for instance, by inhibiting the oxidation of certain oxidizable amino acid residues within the fusion protein, and / or by inhibiting oxidative deamination pathways.

[0285] However, in some embodiments, the pharmaceutical composition is characterized by the absence of antioxidants (or any one or more of those specifically referred to herein with respect to antioxidants).

[0286] Preferably, the antioxidant includes those selected from the group consisting of amino acid or peptide antioxidants, mineral antioxidants, vitamin antioxidants, carotenoid antioxidants, polyphenol antioxidants, aromatic or phenolic antioxidants, chelating antioxidants, thiol antioxidants, and any combination thereof.

[0287] Preferably, the antioxidant comprises an antioxidant consisting of a single compound.

[0288] Preferably, the antioxidant comprises an amino acid antioxidant, such as amino acid antioxidants selected from the group consisting of methionine, N-acetyl-1-cysteine, cysteine, and glutathione.

[0289] The most preferred antioxidant is methionine.

[0290] The pharmaceutical composition preferably contains 0.1-50 mM antioxidant, 1-30 mM antioxidant, 2-20 mM antioxidant, 3-10 mM antioxidant or 4-6 mM antioxidant.

[0291] The pharmaceutical composition preferably contains antioxidants in the following molar ratios of antioxidants to fusion proteins: 910:1 to 1:6, 365:1 to 3:1, 182:1 to 5:1, 100:1 to 7:1, or 50:1 to 10:1.

[0292] The pharmaceutical composition preferably contains 1-30 mM methionine, 2-20 mM methionine, 3-12 mM methionine, 4-6 mM methionine, or about 5 mM methionine.

[0293] The pharmaceutical composition preferably contains methionine in the following molar ratios of methionine to fusion protein: 910:1 to 1:6, 365:1 to 3:1, 182:1 to 5:1, or 100:1 to 7:1, or 50:1 to 10:1.

[0294] The amounts and concentrations related to antioxidants refer to the total amount and concentration of all antioxidants, unless only a single antioxidant is specified. The concentration of the stated or any specific antioxidant can be replaced by a molar ratio.

[0295] Chelating agents

[0296] The pharmaceutical composition may also contain chelating agents. In the context of this invention, chelating agents such as EDTA (and other chelating agents, well known in the art, that may perform similar or identical functions) can increase the stability of the pharmaceutical composition, particularly by inhibiting the degradation of the fusion protein. For example, chelating agents can reduce the likelihood of aggregation and can also act as antioxidants. Chelating agents can also chelate residual metals that would otherwise promote the degradation of the fusion protein and / or other components present in the pharmaceutical composition.

[0297] However, in some embodiments, the pharmaceutical composition is characterized by the absence of a chelating agent (or any one or more of those specifically described herein with respect to chelating agents).

[0298] Preferably, the chelating agent includes EDTA or EGTA, and more preferably, the chelating agent includes EDTA.

[0299] The pharmaceutical composition preferably contains 0.001-0.5 mM chelating agent, 0.01-0.2 mM chelating agent, or 0.025-0.075 mM chelating agent. The pharmaceutical composition preferably contains the chelating agent in the following molar ratios to the fusion protein: 10:1 to 1:550, 4:1 to 1:55, or 1.5:1 to 1:22.

[0300] The pharmaceutical composition preferably contains 0.001-0.5 mM EDTA, 0.01-0.2 mM EDTA, or 0.025-0.075 mM EDTA. The pharmaceutical composition preferably contains EDTA in the following molar ratios to the fusion protein: 10:1 to 1:550, 4:1 to 1:55, or 1.5:1 to 1:22.

[0301] The amounts and concentrations related to chelating agents involve the total amount and concentration of all chelating agents, unless only a single chelating agent is specified. The concentration of the stated or any specific chelating agent can be replaced by a molar ratio.

[0302] weight permeation molar concentration

[0303] Generally, the osmolality of a pharmaceutical composition is not very important; it is determined by the amount of penetrant required to stabilize the formulation. In cases where it is desired to use the pharmaceutical formulation for injection without pre-dilution, such as subcutaneous injection, the formulation is preferably substantially isotonic to avoid skin irritation. In some embodiments, to provide a composition with the desired osmolality, the concentration of the fusion protein can be carefully balanced with the concentrations of a tonic modulator (e.g., NaCl), amino acid components (e.g., arginine / arginine hydrochloride), and sugar components (e.g., trehalose). In some embodiments, the pharmaceutical formulation preferably has an osmolality of 240-640 mOsm / kg, 200-400 mOsm / kg, 250-420 mOsm / kg, 250-350 mOsm / kg, 255-345 mOsm / kg, or 270-310 mOsm / kg. Detailed Implementation

[0305] The embodiments and data presented herein demonstrate that certain technical effects may arise from certain features or combinations of features, and various specific implementations based on these features or combinations of features will be discussed below.

[0306] pH

[0307] Experimental results show that liquid compositions having a pH within 2 pH units, suitably within 1.5 pH units, and suitably even within 1 pH unit of the pI of the TGFβR2 moiety of the fusion protein are unexpectedly stable. Preferably, the pH is greater than or equal to pH 4, more preferably greater than or equal to pH 5. However, high pH can lead to protein stability and solubility problems; therefore, the liquid compositions preferably have a pH less than or equal to pH 7, more preferably less than or equal to pH 6.3. A particularly preferred pH range is pH 5.3–6.3.

[0308] The following numbered paragraphs E1 to E12 disclose specific embodiments of the present invention.

[0309] E1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the pH is within two pH units of the pI of the TGFβR portion of the fusion protein.

[0310] E2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the pH is within 1.5 pH units of the pI of the TGFβR portion of the fusion protein.

[0311] E3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the pH is within one pH unit of the pI of the TGFβR portion of the fusion protein.

[0312] E4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the pH is 4-7.

[0313] E5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the pH is 5.2-6.1.

[0314] E6. The pharmaceutical composition of any one of E1 to E5, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0315] E7. The pharmaceutical composition as described in any one of E1 to E6, further comprising a tension modifier, preferably sodium chloride.

[0316] E8. The pharmaceutical composition as described in any one of E1 to E7, further comprising a surfactant, preferably polysorbate 20.

[0317] E9. The pharmaceutical composition as described in any one of E1 to E8 further comprises a sugar component, preferably trehalose.

[0318] E10. The pharmaceutical composition as described in any one of E1 to E9, further comprising an amino acid component, preferably arginine and / or lysine.

[0319] E11. The pharmaceutical composition as described in any one of E1 to E10 further comprises a buffer system, preferably a histidine buffer system.

[0320] E12. The pharmaceutical composition as described in any one of E1 to E11 further comprises an antioxidant, preferably methionine.

[0321] Aqueous formulations and ionic strength

[0322] Formulation studies revealed specific water-soluble challenges with the IgG:TGFβR fusion protein, suggesting a significant difference in pI between the antibody moiety and the fused TGFβR moiety, rather than being bound by theoretical assumptions. Surprisingly, ionic strength significantly affected the water solubility of the fusion protein. Ionic strength also appears to play a role in protein stability.

[0323] In the early stages of formulation research, lyophilized formulations were considered the only viable option from a long-term storage stability perspective, as described below, which seemed to impose certain limitations. However, as research progressed, liquid formulations were also found to be impractical, but are often preferred for practical reasons (e.g., it avoids energy-intensive lyophilization and manual reconstitution by healthcare professionals).

[0324] As described above in the "Ionic Strength" section, the composition is characterized by its ionic strength, which suitably includes contributions from all ion species in the composition. However, in some embodiments, the composition may be characterized by non-buffered (or tensioner-based) ionic strength, in which case the ionic strength suitably includes contributions from all ion species other than those in the buffer system. Since the buffer system can be used at relatively low concentrations, the following embodiments can be applied to either definition of ionic strength.

[0325] Preferably, the ionic strength of the composition is at least 20 mM, and in the case of a pharmaceutical composition intended for subcutaneous use without prior dilution, its maximum value is preferably controlled by the weight osmolality requirement to avoid any excessive hypertonicity. In a preferred embodiment, the maximum ionic strength is 200 mM, more preferably 160 mM.

[0326] Although ionic strength can be provided by a variety of charged or ionic excipients, it is suitably provided by one or more ionic strain modifiers, such as sodium chloride and / or charged amino acids (e.g., arginine or arginine salts), with sodium chloride being the most preferred.

[0327] Sodium chloride can be the sole non-buffering contributor to ionic strength. In some embodiments, the pharmaceutical composition comprises an amino acid component, which is defined separately from the tonic modifier (e.g., NaCl), and may include, for example, charged or ionic compounds such as arginine or arginine salts. In this case, it should be understood that the amino acid component contributes to the overall ionic strength and the actual tonicity.

[0328] The following numbered paragraphs F1 to F12 disclose specific embodiments of the present invention.

[0329] F1. A pharmaceutical composition comprising IgG:TGFβR fusion protein and characterized in that the ionic strength (optionally unbuffered ionic strength) is greater than or equal to 20 mM.

[0330] F2. A pharmaceutical composition comprising IgG:TGFβR fusion protein, characterized in that the ionic strength (optionally unbuffered ionic strength) is greater than or equal to 20 mM and the weight osmotic molar concentration is 255-345 mOsm / kg.

[0331] F3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized by an ionic strength of 20 to 200 mM (optionally unbuffered ionic strength).

[0332] F4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ion tension modulator of ≥20 mM.

[0333] F5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and sodium chloride at or above 20 mM.

[0334] F6. The pharmaceutical composition as described in any one of F1 to F5, further characterized by the pH as defined in any one of E1 to E5.

[0335] F7. The pharmaceutical composition of any one of F1 to F6, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0336] F8. The pharmaceutical composition as described in any one of F1 to F7, further comprising a surfactant, preferably polysorbate 20.

[0337] F9. The pharmaceutical composition as described in any one of F1 to F8, further comprising a sugar component, preferably trehalose.

[0338] F10. The pharmaceutical composition as described in any one of F1 to F9, further comprising an amino acid component, preferably arginine and / or lysine.

[0339] F11. A pharmaceutical composition of any one of F1 to F10, further comprising a buffer system, preferably a histidine buffer system.

[0340] F12. A pharmaceutical composition of any one of F1 to F11, further comprising an antioxidant, preferably methionine.

[0341] Lyophilization and Ionic Strength

[0342] Early research focused on lyophilized formulations, which were considered the most viable option at the time from a long-term storage perspective. Therefore, a balance was sought between solubility and isotonicity (in liquid form), stability (in lyophilized and reconstituted liquid form), and lyophilizability. According to the "Aqueous Formulations and Ionic Strength" section above, ionic strength, particularly when provided to the composition in the form of sodium chloride, helps meet the requirements for tonicity, stability, and solubility. However, studies have found that excessive sodium chloride usage can affect lyophilization and cause lyophilized cake collapse. While the addition of lyophilization protectants, such as trehalose, can mitigate this to some extent, there appears to be a maximum tolerable amount of sodium chloride to meet the aforementioned objectives of lyophilized formulations. Since higher protein concentrations generally require higher concentrations of sodium chloride, as will be discussed below, this limitation on the amount of sodium chloride also potentially limits the concentration of the fusion protein.

[0343] The following numbered paragraphs G1 to G21 disclose specific embodiments of the present invention.

[0344] G1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized by an ionic strength (optionally unbuffered ionic strength) between 20 and 50 mM.

[0345] G2. A pharmaceutical composition comprising IgG:TGFβR fusion protein, characterized by an ionic strength (optionally unbuffered ionic strength) of 20-50 mM and a weight osmotic molar concentration of 255-345 mOsm / kg.

[0346] G3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 to 50 mM of an ion tension modulator.

[0347] G4. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 20 to 50 mM sodium chloride.

[0348] G5. The pharmaceutical composition as described in any one of G1 to G4, wherein the concentration of the ion tension modifier (or sodium chloride) is 35-45 mM.

[0349] The pharmaceutical composition of any one of G6, G1 to G4, wherein the concentration of the ion tension modifier (or sodium chloride) is about 40 mM.

[0350] G7. The pharmaceutical composition as described in any one of G1 to G6, wherein the concentration of IgG:TGFβR fusion protein is 5-25 mM.

[0351] G8. The pharmaceutical composition as described in any one of G1 to G7, wherein the concentration of the IgG:TGFβR fusion protein is about 10 mM.

[0352] G9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ion tension modulator in a molar ratio of 180:1 to 9110:1 to an ion tension modulator and a fusion protein.

[0353] G10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ion tension modulator in a molar ratio of 360:1 to 911:1 to the ion tension modulator and fusion protein.

[0354] G11. A pharmaceutical composition comprising IgG:TGFβR fusion protein and sodium chloride in the following molar ratio of sodium chloride to fusion protein: 180:1 to 9110:1.

[0355] G12. A pharmaceutical composition comprising IgG:TGFβR fusion protein and sodium chloride in the following molar ratio of sodium chloride to fusion protein: 360:1 to 911:1.

[0356] G13. The pharmaceutical composition as described in any one of G1 to G12, further characterized by a pH as defined in any one of E1 to E5.

[0357] G14. The pharmaceutical composition as described in any one of G1 to G13, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0358] G15. The pharmaceutical composition as described in any one of G1 to G14, further comprising a surfactant, preferably polysorbate 20.

[0359] G16. The pharmaceutical composition as described in any one of G1 to G15 further comprises a sugar component, preferably trehalose.

[0360] G17. The pharmaceutical composition as described in any one of G1 to G16 further comprises an amino acid component, preferably arginine and / or lysine.

[0361] G18. The pharmaceutical composition as described in any one of G1 to G17 further comprises a buffer system, preferably a histidine buffer system.

[0362] G19. The pharmaceutical composition as described in any one of G1 to G18 further comprises an antioxidant, preferably methionine.

[0363] G20. A pharmaceutical composition as described in any one of G1 to G19, wherein the composition is a reconstituted lyophilized pharmaceutical composition.

[0364] G21. The pharmaceutical composition as described in any one of G1 to G19, wherein it is a solid lyophilized pharmaceutical composition suitable for reconstruction.

[0365] Tension-regulating lyophilization protectant

[0366] In the case of lyophilized formulations, the addition of tension-modifying lyophilizers has been found to help achieve a reasonable balance between solubility and isotonicity (liquid form), stability (lyophilized and reconstituted liquid form), and lyophilizability. Tension-modifying lyophilizers can provide tension without compromising lyophilizability, unlike ionic strength providers (such as sodium chloride), which can damage the lyophilized cake. Therefore, minimal but sufficiently high amounts of ionic strength providers (such as NaCl) are permitted to promote protein solubility and stability. In many cases, the use of tension-modifying lyophilizers can at least partially offset the negative effects of ionic tension modifiers (such as NaCl) on lyophilization.

[0367] Suitable tension-regulating lyophilization protectants include sugar components and / or amino acid components, such as trehalose and / or arginine, but more preferably sugar components (preferably a single sugar component). Preferred sugar components are described herein, although trehalose is the most suitable sugar component.

[0368] The following numbered paragraphs H1 to H34 disclose specific embodiments of the present invention.

[0369] H1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component as defined above), and an ion tension modulator.

[0370] H2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component as defined above), and an ion tension modulator, wherein the molar ratio of the tension-modulating lyophilization protectant to the ion tension modulator is 10:1 to 1:2.

[0371] H3. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and sodium chloride in a molar ratio of trehalose to sodium chloride of 10:1 to 1:2.

[0372] H4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component as defined above) and 20 to 50 mM of an ionic tension modulator.

[0373] H5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component), and 20 to 50 mM sodium chloride.

[0374] H6. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and 20 to 50 mM of an ion tension modulator.

[0375] H7. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and 20 to 50 mM sodium chloride.

[0376] H8. The pharmaceutical composition as described in any one of H1 to H7, wherein the tension-modifying lyophilization protectant is present at a concentration of 100-200 mM, preferably 130-170 mM.

[0377] H9. A pharmaceutical composition of any one of H1 to H8, wherein the concentration of IgG:TGFβR fusion protein is 5-25 mM.

[0378] H10. The pharmaceutical composition as described in any one of H1 to H9, wherein the concentration of the IgG:TGFβR fusion protein is about 10 mM.

[0379] H11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a glycosylation component and / or an amino acid component), and an ion tension modulator, wherein the molar ratio of the ion tension modulator to the fusion protein is from 180:1 to 9110:1.

[0380] H12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a glycosylation component and / or an amino acid component), and an ion tension modulator, wherein the molar ratio of the ion tension modulator to the fusion protein is from 360:1 to 911:1.

[0381] H13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component), and sodium chloride, wherein the molar ratio of sodium chloride to the fusion protein is from 180:1 to 9110:1.

[0382] H14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a tension-modulating lyophilization protectant (e.g., a sugar component and / or an amino acid component), and sodium chloride, wherein the molar ratio of sodium chloride to the fusion protein is from 360:1 to 911:1.

[0383] H15. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and an ion tension modifier in a molar ratio of 180:1 to 9110:1 to the ion tension modifier and the fusion protein.

[0384] H16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and an ion tension modifier in a molar ratio of 360:1 to 911:1 to the ion tension modifier and the fusion protein.

[0385] H17. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and sodium chloride in a molar ratio of sodium chloride to fusion protein of 180:1 to 9110:1.

[0386] H18. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and sodium chloride in a molar ratio of sodium chloride to fusion protein of 360:1 to 911:1.

[0387] H19. The pharmaceutical composition of any one of H1 to H18, wherein the tension-modulating lyophilization protectant is selected from the group consisting of trehalose, sucrose, sorbitol, mannitol, arginine, and any combination thereof.

[0388] H20. A pharmaceutical composition of any one of H1 to H19, wherein the tension-regulating lyophilization protectant is a combination of trehalose and arginine.

[0389] H21. The pharmaceutical composition as described in any one of H1 to H20, wherein the tension-regulating lyophilization protectant is trehalose only.

[0390] H22. The pharmaceutical composition as described in any one of H1 to H21, wherein the molar ratio of the tension-modifying lyophilization protectant to the ion tension modifier to the fusion protein is 910-36430:180-9110:1.

[0391] A pharmaceutical composition of any one of H23, H1 to H22, wherein the molar ratio of the tension-modifying lyophilization protectant to the ion tension modifier to the fusion protein is 1820-3643:360-911:1.

[0392] H24. The pharmaceutical composition as described in any one of H1 to H23, wherein the molar ratio of trehalose to sodium chloride to the fusion protein is 1820-3643:360-911:1.

[0393] H25. A pharmaceutical composition as described in any one of H1 to H24, further characterized by a pH as defined in any one of E1 to E5.

[0394] H26. The pharmaceutical composition of any one of H1 to H25, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0395] H27. The pharmaceutical composition as described in any one of H1 to H26, further comprising a surfactant, preferably polysorbate 20.

[0396] H28. The pharmaceutical composition as described in any one of H1 to H27, further comprising a sugar component, preferably trehalose.

[0397] H29. The pharmaceutical composition as described in any one of H1 to H28 further comprises an amino acid component, preferably arginine and / or lysine.

[0398] H30. The pharmaceutical composition as described in any one of H1 to H29 further comprises a buffer system, preferably a histidine buffer system.

[0399] H31. The pharmaceutical composition as described in any one of H1 to H30 further comprises an antioxidant, preferably methionine.

[0400] H32. The pharmaceutical composition as described in any one of H1 to H31, wherein it is a reconstituted lyophilized pharmaceutical composition.

[0401] H33. The pharmaceutical composition as described in any one of H1 to H31, which is a solid lyophilized pharmaceutical composition suitable for reconstruction.

[0402] H34. The pharmaceutical composition of any one of H1 to H33, wherein the ion tension modifier is a non-buffered ion tension modifier.

[0403] pH and ionic strength

[0404] As mentioned earlier, ionic strength (especially when provided by NaCl) can improve the solubility of fusion proteins at all pH levels, but this effect is particularly significant at higher pH levels.

[0405] The following numbered paragraphs I1 to I14 disclose specific embodiments of the present invention.

[0406] 11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ionic tonic modulator of ≥20 mM, characterized in that the pH is 5-7.

[0407] 12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of sodium chloride, characterized in that the pH is 5-7.

[0408] 13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ionic tonic modulator of ≥20 mM, characterized in that the pH is 5.4-6.4.

[0409] 14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of sodium chloride, characterized in that the pH is 5.4-6.4.

[0410] 15. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ionic tonic modulator of ≥20 mM, characterized in that the pH is 5.8-6.8.

[0411] 16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of sodium chloride, characterized in that the pH is 5.8-6.8.

[0412] 17. The pharmaceutical composition of any one of 11 to 16, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0413] 18. The pharmaceutical composition as described in any one of 11 to 17, further comprising a surfactant, preferably polysorbate 20.

[0414] 19. The pharmaceutical composition as described in any one of 11 to 18 further comprises a sugar component, preferably trehalose.

[0415] I10. The pharmaceutical composition as described in any one of I1 to I9, further comprising an amino acid component, preferably arginine and / or lysine.

[0416] I11. The pharmaceutical composition as described in any one of I1 to I10 further comprises a buffer system, preferably a histidine buffer system.

[0417] I12. The pharmaceutical composition as described in any one of I1 to I11, further comprising an antioxidant, preferably methionine.

[0418] I13. The pharmaceutical composition as described in any one of I1 to I12, wherein it is a liquid pharmaceutical composition.

[0419] I14. The pharmaceutical composition of any one of I1 to I13, wherein the ion tension modifier is a non-buffered ion tension modifier.

[0420] Fusion protein concentration

[0421] Although early studies suggested that the concentration of fusion proteins in lyophilized formulations might be limited by the maximum amount of sodium chloride that the lyophilization process could tolerate, later studies demonstrated the long-term viability of liquid formulations, which further unlocked the potential for higher fusion protein concentrations, especially with appropriate support of increased ionic strength.

[0422] The following numbered paragraphs J1 to J12 disclose specific embodiments of the present invention.

[0423] J1. A pharmaceutical composition comprising more than 20 mg / mL, preferably more than 80 mg / mL, of IgG:TGFβR fusion protein.

[0424] J2. A pharmaceutical composition comprising more than 20 mg / mL, preferably more than 80 mg / mL, of IgG:TGFβR fusion protein, and more than or equal to 20 mM of an ionic tonic modulator.

[0425] J3. A pharmaceutical composition comprising more than 20 mg / mL, preferably more than 80 mg / mL, of IgG:TGFβR fusion protein and more than or equal to 20 mM of an ionic tonic modulator, characterized in that the pH is 4-8.

[0426] J4. The pharmaceutical composition as described in any one of J1 to J3, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0427] J5. The pharmaceutical composition as described in any one of J1 to J4, characterized by a pH as defined in any one of E1 to E5.

[0428] J6. The pharmaceutical composition as described in any one of J1 to J5 further comprises a tension modifier, preferably sodium chloride.

[0429] J7. The pharmaceutical composition as described in any one of J1 to J6, further comprising a surfactant, preferably polysorbate 20.

[0430] J8. The pharmaceutical composition as described in any one of J1 to J7, further comprising a sugar component, preferably trehalose.

[0431] J9. The pharmaceutical composition as described in any one of J1 to J8, further comprising an amino acid component, preferably arginine and / or lysine.

[0432] J10. The pharmaceutical composition as described in any one of J1 to J9 further comprises a buffer system, preferably a histidine buffer system.

[0433] J11. The pharmaceutical composition as described in any one of J1 to J10 further comprises an antioxidant, preferably methionine.

[0434] J12. The pharmaceutical composition as described in any one of J1 to J11, wherein it is a liquid pharmaceutical composition.

[0435] Fusion protein concentration and ionic strength

[0436] Studies have shown that higher concentrations of fusion proteins can be achieved, but this is preferably supported by increasing ionic strength. Increasing ionic strength (especially sodium chloride concentration) in conjunction with fusion protein concentration ensures higher protein solubility and stability. In some cases, sugar components (e.g., trehalose) have been found to partially compensate for the lack of NaCl; the presence of ionic strength providers is generally preferred.

[0437] The following numbered paragraphs K1 to K27 disclose specific embodiments of the present invention.

[0438] K1. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 5-250 mM (optionally unbuffered ionic strength).

[0439] K2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the ionic strength is in the molar ratio of ionic strength to fusion protein of 1000:1 to 70:1.

[0440] K3. A pharmaceutical composition comprising 5-25 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 20-50 mM (optionally unbuffered ionic strength).

[0441] K4. A pharmaceutical composition comprising 25-45 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 40-80 mM (optionally unbuffered ionic strength).

[0442] K5. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of at least 50 mM (optionally unbuffered ionic strength).

[0443] K6. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 80-170 mM (optionally unbuffered ionic strength).

[0444] K7. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 80-120 mM (optionally unbuffered ionic strength), preferably wherein a sugar component (most preferably trehalose) is also present.

[0445] K8. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 120-170 mM (optionally unbuffered ionic strength), preferably free of sugar components (most preferably trehalose).

[0446] K9. A pharmaceutical composition comprising 65-115 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of 100-200 mM (optionally unbuffered ionic strength).

[0447] K10. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein and 10-200 mM tension modulator (preferably 10-200 mM sodium chloride).

[0448] K11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a tension modulator (preferably sodium chloride), wherein the molar ratio of the tension modulator (preferably sodium chloride) to the fusion protein is 910:1 to 200:1.

[0449] K12. A pharmaceutical composition comprising 5-25 mg / mL IgG:TGFβR fusion protein and 20-50 mM tension modulator (preferably 20-50 mM sodium chloride).

[0450] K13. A pharmaceutical composition comprising 25-45 mg / mL IgG:TGFβR fusion protein and 40-80 mM tension modulator (preferably 40-80 mM sodium chloride).

[0451] K14. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein and at least 50 mM tension modulator (preferably at least 50 mM sodium chloride).

[0452] K15. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 80-170 mM tension modulator (preferably 80-170 mM sodium chloride).

[0453] K16. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 80-120 mM tonic modulator (preferably 80-120 mM sodium chloride), preferably also containing a sugar component (most preferably trehalose).

[0454] K17. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 120-170 mM tonic modulator (preferably 120-170 mM sodium chloride), preferably without any sugar component (most preferably trehalose).

[0455] K18. A pharmaceutical composition comprising 65-115 mg / mL IgG:TGFβR fusion protein and 100-200 mM tonic modulator (preferably 100-200 mM sodium chloride).

[0456] K19. The pharmaceutical composition as described in any one of K1 to K18, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0457] K20. The pharmaceutical composition as described in any one of K1 to K19, characterized by a pH as defined in any one of E1 to E5.

[0458] K21. The pharmaceutical composition as described in any one of K1 to K20, further comprising a tension modifier, preferably sodium chloride.

[0459] K22. The pharmaceutical composition as described in any one of K1 to K21, further comprising a surfactant, preferably polysorbate 20.

[0460] K23. The pharmaceutical composition as described in any one of K1 to K22 further comprises a sugar component, preferably trehalose.

[0461] K24. The pharmaceutical composition as described in any one of K1 to K23, further comprising an amino acid component, preferably arginine and / or lysine.

[0462] K25. The pharmaceutical composition as described in any one of K1 to K24, further comprising a buffer system, preferably a histidine buffer system.

[0463] K26. The pharmaceutical composition as described in any one of K1 to K25, further comprising an antioxidant, preferably methionine.

[0464] K27. The pharmaceutical composition as described in any one of K1 to K26, wherein it is a liquid pharmaceutical composition.

[0465] Fusion protein concentration and pH

[0466] It has been found that, in general, higher concentrations of fusion proteins tend to be better supported by slightly higher pH values, although the pH is still closer to the pI of the TGFβR portion than is normally expected.

[0467] The following numbered paragraphs L1 to L17 disclose specific embodiments of the present invention.

[0468] L1. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is 5.0-7.5, preferably 5.0-7.0.

[0469] L2. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein, characterized by a pH of 5.3-6.3, and further characterized by the absence of acetate buffer and acetate.

[0470] L3. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein and a histidine buffer system, characterized in that the pH is pH 5.3-6.3.

[0471] L4. A pharmaceutical composition comprising 5-25 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is pH 5.0-6.5, preferably pH 5.0-5.7.

[0472] L5. A pharmaceutical composition comprising 25-45 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is 5.0-7.5, preferably 5.3-6.2.

[0473] L6. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is pH 5.0-7.5, preferably pH 5.3-6.5.

[0474] L7. A pharmaceutical composition comprising at least 35 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is pH 5.0-7.5, preferably pH 5.4-7.0.

[0475] L8. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is 5.0-7.5, preferably 5.5-6.5.

[0476] L9. A pharmaceutical composition comprising 65-115 mg / mL IgG:TGFβR fusion protein, characterized in that the pH is pH 5.0-8.0, preferably pH 5.8-6.8.

[0477] L10. The pharmaceutical composition as described in any one of L1 to L9, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0478] L11. The pharmaceutical composition as described in any one of L1 to L10 further comprises a tension modifier, preferably sodium chloride.

[0479] L12. The pharmaceutical composition as described in any one of L1 to L11, further comprising a surfactant, preferably polysorbate 20.

[0480] L13. The pharmaceutical composition as described in any one of L1 to L12 further comprises a sugar component, preferably trehalose.

[0481] L14. The pharmaceutical composition as described in any one of L1 to L13, further comprising an amino acid component, preferably arginine and / or lysine.

[0482] L15. A pharmaceutical composition of any one of L1 to L14, further comprising a buffer system, preferably a histidine buffer system.

[0483] L16. A pharmaceutical composition of any one of L1 to L15, further comprising an antioxidant, preferably methionine.

[0484] L17. The pharmaceutical composition as described in any one of L1 to L16, wherein it is a liquid pharmaceutical composition.

[0485] Fusion protein concentration, ionic strength and pH

[0486] As mentioned above, higher concentrations of fusion proteins are often best supported by slightly higher pH and slightly higher ionic strength.

[0487] The following numbered paragraphs M1 to M28 disclose specific embodiments of the present invention.

[0488] M1. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength (optionally unbuffered ionic strength) of 5-250 mM and a pH of pH 5.0-7.0.

[0489] M2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, characterized in that the molar ratio of ionic strength to fusion protein is 1000:1 to 70:1 and the pH is pH 5.0-6.5.

[0490] M3. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein, characterized in that the molar ratio of ionic strength to fusion protein is 375:1 to 150:1, and the pH is pH 5.3-6.3.

[0491] M4. A pharmaceutical composition comprising 5-25 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 20-50 mM (optionally unbuffered ionic strength) and a pH of pH 5.0-6.5, preferably pH 5.0-5.7.

[0492] M5. A pharmaceutical composition comprising 25-45 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 40-80 mM (optionally unbuffered ionic strength) and a pH of pH 5.0-7.5, preferably pH 5.3-6.2.

[0493] M6. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein, characterized in that it has an ionic strength of at least 50 mM (optionally unbuffered ionic strength) and a pH of pH 5.0-7.5, preferably pH 5.3-6.5.

[0494] M7. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 80-170 mM (optionally unbuffered ionic strength) and a pH of 5.0-7.5, preferably 5.5-6.5.

[0495] M8. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 80-120 mM (optionally unbuffered ionic strength) and a pH of pH 5.0-7.5, preferably pH 5.5-6.5, and preferably also containing a sugar component (most preferably trehalose).

[0496] M9. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength of 120-170 mM (optionally unbuffered ionic strength), a pH of pH 5.0-7.5, preferably pH 5.5-6.5, and preferably in the absence of a sugar component (most preferably trehalose).

[0497] M10. A pharmaceutical composition comprising 65-115 mg / mL IgG:TGFβR fusion protein, characterized by an ionic strength (optionally unbuffered ionic strength) of 100-200 mM and a pH of pH 5.0-8.0, preferably pH 5.8-6.8.

[0498] M11. A pharmaceutical composition comprising 5-150 mg / mL IgG:TGFβR fusion protein and 10-200 mM tension modulator (preferably 10-200 mM sodium chloride), characterized in that the pH is pH 5-7.

[0499] M12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a tension modulator (preferably sodium chloride), wherein the molar ratio of the tension modulator (preferably sodium chloride) to the fusion protein is 910:1 to 200:1, and further characterized in that the pH is pH 5-7.0.

[0500] M13. A pharmaceutical composition comprising 5-25 mg / mL IgG:TGFβR fusion protein and 20-50 mM tension modulator (preferably 20-50 mM sodium chloride), further characterized in that the pH is 5.0-6.5, preferably 5.0-5.7.

[0501] M14. A pharmaceutical composition comprising 25-45 mg / mL IgG:TGFβR fusion protein and 40-80 mM tonic modulator (preferably 40-80 mM sodium chloride), characterized in that the pH is 5.0-7.5, preferably 5.3-6.2.

[0502] M15. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein and at least 50 mM tension modulator (preferably at least 50 mM sodium chloride), characterized in that the pH is pH 5.0-7.5, preferably pH 5.3-6.5.

[0503] M16. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 80-170 mM tension modulator (preferably 80-170 mM sodium chloride), characterized in that the pH is 5.0-7.5, preferably 5.5-6.5.

[0504] M17. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 80-120 mM tonic modulator (preferably 80-120 mM sodium chloride), further characterized in that the pH is 5.0-7.5, preferably 5.5-6.5, and preferably also contains a sugar component (most preferably trehalose).

[0505] M18. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 120-170 mM tonic modulator (preferably 120-170 mM sodium chloride), characterized in that the pH is 5.0-7.5, preferably 5.5-6.5, and preferably in the absence of a sugar component (most preferably trehalose).

[0506] M19. A pharmaceutical composition comprising 65-115 mg / mL IgG:TGFβR fusion protein and 100-200 mM tension modulator (preferably 100-200 mM sodium chloride), characterized in that the pH is 5.0-8.0, preferably 5.8-6.8.

[0507] M20. The pharmaceutical composition as described in any one of M1 to M19, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0508] M21. The pharmaceutical composition as described in any one of M1 to M20 further comprises a tension modifier, preferably sodium chloride.

[0509] M22. The pharmaceutical composition as described in any one of M1 to M21, further comprising a surfactant, preferably polysorbate 20.

[0510] M23. The pharmaceutical composition as described in any one of M1 to M22 further comprises a sugar component, preferably trehalose.

[0511] M24. The pharmaceutical composition as described in any one of M1 to M23, further comprising an amino acid component, preferably arginine and / or lysine.

[0512] M25. The pharmaceutical composition as described in any one of M1 to M24, further comprising a buffer system, preferably a histidine buffer system.

[0513] M26. The pharmaceutical composition as described in any one of M1 to M25, further comprising an antioxidant, preferably methionine.

[0514] M27. The pharmaceutical composition as described in any one of M1 to M26, further characterized in that it is free of acetate buffer and acetate.

[0515] M28. The pharmaceutical composition as described in any one of M1 to M27, wherein it is a liquid pharmaceutical composition.

[0516] Sugar components

[0517] Experimental studies generally indicate that the inclusion of sugar components is advantageous, especially sugar components selected from non-reducing disaccharides and non-reducing sugar polyols, among which trehalose appears to be superior to all other sugar components.

[0518] The following numbered paragraphs N1 to N24 disclose specific embodiments of the present invention.

[0519] N1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a glycoside.

[0520] N2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 30-300 mM glycoside.

[0521] N3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 40-110 mM glycoside.

[0522] N4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 100-200 mM glycoside.

[0523] N5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar component in a molar ratio of 3700:1 to 70:1 for the sugar component to the fusion protein.

[0524] N6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol.

[0525] N7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 30-300 mM glycoside selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol.

[0526] N8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 40-110 mM glycoside selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol.

[0527] N9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 100-200 mM of a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol.

[0528] N10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, wherein the molar ratio of the sugar component to the fusion protein is from 3700:1 to 70:1.

[0529] N11. A pharmaceutical composition comprising IgG:TGFβR fusion protein and trehalose.

[0530] N12. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 30-300 mM trehalose.

[0531] N13. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 40-110 mM trehalose.

[0532] N14. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 100-200 mM trehalose.

[0533] N15. A pharmaceutical composition comprising IgG:TGFβR fusion protein and trehalose in a molar ratio of 3700:1 to 70:1 for trehalose to fusion protein.

[0534] N16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a disaccharide.

[0535] N17. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 40-200 mM disaccharide.

[0536] N18. The pharmaceutical composition of any one of N1 to N17, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9 and M1 to M19.

[0537] N19. The pharmaceutical composition of any one of N1 to N18, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0538] N20. The pharmaceutical composition as described in any one of N1 to N19, further comprising a tension modifier, preferably sodium chloride.

[0539] N21. The pharmaceutical composition as described in any one of N1 to N20, further comprising a surfactant, preferably polysorbate 20.

[0540] N22. The pharmaceutical composition of any one of N1 to N21 further comprises an amino acid component, preferably arginine and / or lysine.

[0541] N23. ​​The pharmaceutical composition as described in any one of N1 to N22, further comprising a buffer system, preferably a histidine buffer system.

[0542] N24. The pharmaceutical composition as described in any one of N1 to N23, further comprising an antioxidant, preferably methionine.

[0543] amino acid components

[0544] Experimental studies generally show that the inclusion of amino acid components is advantageous, especially those selected from the following group: arginine, lysine, proline, glutamic acid, and glycine, among which arginine and, to some extent, lysine appear to be superior to all other amino acid components.

[0545] The following numbered paragraphs O1 to O20 disclose specific embodiments of the present invention.

[0546] O1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component.

[0547] O2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 10-300 mM amino acid component.

[0548] O3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 30-110 mM amino acid component.

[0549] O4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component in a molar ratio of 5500:1 to 18:1 with the following amino acid components to the fusion protein.

[0550] O5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof.

[0551] O6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 10-300 mM amino acid component, said amino acid component being selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof.

[0552] O7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a 30-110 mM amino acid component, said amino acid component being selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof.

[0553] O8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof, in a molar ratio of 5500:1 to 18:1.

[0554] O9. A pharmaceutical composition comprising IgG:TGFβR fusion protein and arginine.

[0555] O10. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 10-300 mM arginine.

[0556] O11. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 30-110 mM arginine.

[0557] O12. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 30-80 mM arginine.

[0558] O13. A pharmaceutical composition comprising IgG:TGFβR fusion protein and arginine in the following molar ratio of arginine to fusion protein: 5500:1 to 18:1.

[0559] O14. The pharmaceutical composition as described in any one of O1 to O13, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9, M1 to M19 and N1 to N17.

[0560] O15. The pharmaceutical composition of any one of O1 to O14, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0561] O16. The pharmaceutical composition as described in any one of O1 to O15, further comprising a tension modifier, preferably sodium chloride.

[0562] O17. The pharmaceutical composition as described in any one of O1 to O16, further comprising a surfactant, preferably polysorbate 20.

[0563] O18. The pharmaceutical composition as described in any one of O1 to O17 further comprises a sugar component, preferably trehalose.

[0564] O19. The pharmaceutical composition as described in any one of O1 to O18 further comprises a buffer system, preferably a histidine buffer system.

[0565] O20. The pharmaceutical composition as described in any one of O1 to O19 further comprises an antioxidant, preferably methionine.

[0566] Sugar components and amino acid components

[0567] Studies have shown that arginine, trehalose, and lysine all contribute to formulation stability. Although arginine could potentially replace or at least partially replace trehalose, the combined use of trehalose and arginine has been found to be particularly stable.

[0568] The following numbered paragraphs P1 to P18 disclose specific embodiments of the present invention.

[0569] P1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a glycosylation component, and an amino acid component.

[0570] P2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a 30-300 mM glycosylation component, and a 10-300 mM amino acid component.

[0571] P3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a 40-110 mM glycosylation component, and a 30-110 mM amino acid component.

[0572] P4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein in a molar ratio of 3700:1 to 70:1 for the sugar component and the fusion protein, and further comprising an amino acid component in a molar ratio of 5500:1 to 18:1 for the amino acid component and the fusion protein.

[0573] P5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a glycoside selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine.

[0574] P6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 30-300 mM of a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, and 10-300 mM of an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine.

[0575] P7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 40-110 mM of a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, and 30-110 mM of an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine.

[0576] P8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, wherein the molar ratio of the sugar component to the fusion protein is from 3700:1 to 70:1, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine, wherein the molar ratio of the amino acid component to the fusion protein is from 5500:1 to 18:1.

[0577] P9. A pharmaceutical composition comprising IgG:TGFβR fusion protein, trehalose, and arginine.

[0578] P10. A pharmaceutical composition comprising IgG:TGFβR fusion protein, 30-300 mM trehalose and 10-300 mM arginine.

[0579] P11. A pharmaceutical composition comprising IgG:TGFβR fusion protein, 40-110 mM trehalose and 30-110 mM arginine.

[0580] P12. A pharmaceutical composition comprising IgG:TGFβR fusion protein and trehalose in a molar ratio of 3700:1 to 70:1, and arginine in a molar ratio of 5500:1 to 18:1.

[0581] P13. The pharmaceutical composition as described in any one of P1 to P12, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9 and M1 to M19.

[0582] P14. The pharmaceutical composition as described in any one of P1 to P13, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0583] P15. The pharmaceutical composition as described in any one of P1 to P14 further comprises a tension modifier, preferably sodium chloride.

[0584] P16. The pharmaceutical composition as described in any one of P1 to P15 further comprises a surfactant, preferably polysorbate 20.

[0585] P17. The pharmaceutical composition as described in any one of P1 to P16 further comprises a buffer system, preferably a histidine buffer system.

[0586] P18. The pharmaceutical composition as described in any one of P1 to P17 further comprises an antioxidant, preferably methionine.

[0587] Sugar and amino acid components as substitutes for sodium chloride

[0588] Later studies have shown that arginine, and to some extent trehalose, may potentially replace or partially replace NaCl as a source of ionic strength.

[0589] The following numbered paragraphs Q1 to Q17 disclose specific embodiments of the present invention.

[0590] Q1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, a sugar component, and an amino acid component.

[0591] Q2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride and amino acid components.

[0592] Q3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, a sugar component, and an amino acid component; wherein the total molar concentration of the combination of one or more of the sodium chloride, sugar component, and amino acid component is 150 mM to 250 mM.

[0593] Q4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, a sugar component, and an amino acid component; wherein the total molar concentration of the combination of one or more of the sodium chloride, sugar component, and amino acid component is from 180 mM to 220 mM.

[0594] Q5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of the following: sodium chloride, a sugar component selected from the group consisting of trehalose, sucrose, mannitol and sorbitol, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid and glycine.

[0595] Q6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, sodium chloride, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine.

[0596] Q7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of the following: sodium chloride, a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine; wherein the total molar concentration of the combination of one or more of the sodium chloride, sugar component, and amino acid component is from 150 mM to 250 mM.

[0597] Q8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of the following: sodium chloride, a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine; wherein the total molar concentration of the combination of one or more of the sodium chloride, sugar component, and amino acid component is from 180 mM to 220 mM.

[0598] Q9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, trehalose, and arginine.

[0599] Q10. A pharmaceutical composition comprising IgG:TGFβR fusion protein, sodium chloride, and arginine.

[0600] Q11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, trehalose, and arginine; wherein the total molar concentration of the combination of one or more of sodium chloride, trehalose, and arginine is 150 mM to 250 mM.

[0601] Q12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, and one or more of sodium chloride, trehalose, and arginine; wherein the total molar concentration of the combination of one or more of sodium chloride, trehalose, and arginine is from 180 mM to 220 mM.

[0602] Q13. The pharmaceutical composition as described in any one of Q1 to Q12, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9, M1 to M19 and N1 to N17.

[0603] Q14. The pharmaceutical composition of any one of Q1 to Q13, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0604] Q15. The pharmaceutical composition as described in any one of Q1 to Q14, further comprising a surfactant, preferably polysorbate 20.

[0605] Q16. A pharmaceutical composition of any one of Q1 to Q15, further comprising a buffer system, preferably a histidine buffer system.

[0606] Q17. A pharmaceutical composition of any one of Q1 to Q16, further comprising an antioxidant, preferably methionine.

[0607] surfactants

[0608] Studies have shown that surfactants are desirable, with polysorbate surfactants, especially polysorbate 20, performing best. Surfactants are particularly useful in mitigating aggregation.

[0609] The following numbered paragraphs R1 to R34 disclose specific embodiments of the present invention.

[0610] R1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant.

[0611] R2. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.01-2 mg / mL surfactant.

[0612] R3. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.05-1.5 mg / mL surfactant.

[0613] R4. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.4-1.2 mg / mL surfactant.

[0614] R5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4-0.6 mg / mL of surfactant.

[0615] R6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and about 0.5 mg / mL of surfactant.

[0616] R7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant in a surfactant-fusion protein molar ratio of 30:1 to 1:70.

[0617] R8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant in a surfactant-to-fusion protein molar ratio of 12:1 to 1:3.

[0618] R9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant in a molar ratio of 8:1 to 1:2 to the surfactant and the fusion protein.

[0619] R10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80.

[0620] R11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 at a concentration of 0.01-2 mg / mL.

[0621] R12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 at a concentration of 0.05-1.5 mg / mL.

[0622] R13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 at a concentration of 0.4-1.2 mg / mL.

[0623] R14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 at a concentration of 0.4-0.6 mg / mL.

[0624] R15. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and about 0.5 mg / mL of a surfactant selected from polysorbate 20 or polysorbate 80.

[0625] R16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 in a molar ratio of 30:1 to 1:70 to the surfactant and the fusion protein.

[0626] R17. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 in a molar ratio of 12:1 to 1:3 to the surfactant and the fusion protein.

[0627] R18. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80 in a molar ratio of 8:1 to 1:2 to the surfactant and the fusion protein.

[0628] R19. A pharmaceutical composition comprising IgG:TGFβR fusion protein and polysorbate 20.

[0629] R20. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.01-2 mg / mL polysorbate 20.

[0630] R21. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.05-1.5 mg / mL polysorbate 20.

[0631] R22. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.4-1.2 mg / mL polysorbate 20.

[0632] R23. A pharmaceutical composition comprising IgG:TGFβR fusion protein and 0.4-0.6 mg / mL polysorbate 20.

[0633] R24. A pharmaceutical composition comprising IgG:TGFβR fusion protein and about 0.5 mg / mL polysorbate 20.

[0634] R25. A pharmaceutical composition comprising IgG:TGFβR fusion protein and polysorbate 20 in a molar ratio of 30:1 to 1:70 of polysorbate 20 to fusion protein.

[0635] R26. A pharmaceutical composition comprising IgG:TGFβR fusion protein and polysorbate 20 in a molar ratio of 12:1 to 1:3 of polysorbate 20 to fusion protein.

[0636] R27. A pharmaceutical composition comprising IgG:TGFβR fusion protein and polysorbate 20 in a molar ratio of 8:1 to 1:2 of polysorbate 20 to fusion protein.

[0637] R28. The pharmaceutical composition as described in any one of R1 to R27, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9, M1 to M19, N1 to N17, O1 to O13, P1 to P12 and Q1 to Q12.

[0638] R29. The pharmaceutical composition of any one of R1 to R28, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0639] R30. The pharmaceutical composition as described in any one of R1 to R29, further comprising a tension modifier, preferably sodium chloride.

[0640] R31. The pharmaceutical composition as described in any one of R1 to R30, further comprising a sugar component, preferably trehalose.

[0641] R32. The pharmaceutical composition as described in any one of R1 to R31, further comprising an amino acid component, preferably arginine and / or lysine.

[0642] R33. A pharmaceutical composition of any one of R1 to R32, further comprising a buffer system, preferably a histidine buffer system.

[0643] R34. A pharmaceutical composition of any one of R1 to R33, further comprising an antioxidant, preferably methionine.

[0644] buffer

[0645] Since compositions without buffers appear to be less preferred than those containing buffers, the pharmaceutical compositions of the present invention preferably contain a buffering system. Although many buffering systems can be used to buffer the pharmaceutical compositions of the present invention at a suitable pH (suitably as defined herein), some buffers appear to be preferred over others. However, studies have shown that the buffer concentration appears to be less important.

[0646] Overall, histidine buffering systems performed best. Although acetate buffers can be used, they should be appropriately excluded due to potential skin irritation. Similarly, citrate buffers are advantageously excluded from the pharmaceutical compositions of the present invention, although citrate buffers can be used at higher pH levels.

[0647] The following numbered paragraphs S1 to S18 disclose specific embodiments of the present invention.

[0648] S1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system.

[0649] S2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system selected from the group consisting of histidine buffer, phosphate buffer, succinate buffer, citrate buffer, and any combination thereof.

[0650] S3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system selected from the group consisting of histidine buffer, phosphate buffer, succinate buffer, and citrate buffer.

[0651] S4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a histidine buffer system.

[0652] S5. The pharmaceutical composition as described in any one of S1 to S4, wherein the buffer system is present at a concentration of 2-70 mM.

[0653] S6. The pharmaceutical composition as described in any one of S1 to S4, wherein the buffer system is present at a concentration of 4-30 mM.

[0654] S7. The pharmaceutical composition as described in any one of S1 to S4, wherein the buffer system is present at a concentration of 5-15 mM.

[0655] S8. The pharmaceutical composition as described in any one of S1 to S4, wherein the buffer system is present in a molar ratio of the buffer system to the fusion protein of 1280:1 to 3:1.

[0656] S9. The pharmaceutical composition of any one of S1 to S4, wherein the buffer system is present in a molar ratio of the buffer system to the fusion protein of 370:1 to 9:1.

[0657] S10. The pharmaceutical composition of any one of S1 to S4, wherein the buffer system is present in a molar ratio of the buffer system to the fusion protein of 100:1 to 15:1.

[0658] S11. The pharmaceutical composition as described in any one of S1 to S4; wherein the composition is further characterized in that it is free of acetate buffer and acetate.

[0659] S12. The pharmaceutical composition as described in any one of S1 to S11, further characterized by any one of E1 to E5, F1 to F5, G1 to G12, H1 to H24, I1 to I6, J1 to J4, K1 to K18, L1 to L9, M1 to M19, N1 to N17, O1 to O13, P1 to P12, Q1 to Q12, and R1 to R27.

[0660] S13. The pharmaceutical composition as described in any one of S1 to S12, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having the same amino acid sequence as bintrafusp alfa.

[0661] S14. The pharmaceutical composition as described in any one of S1 to S13 further comprises a tension modifier, preferably sodium chloride.

[0662] S15. The pharmaceutical composition as described in any one of S1 to S14 further comprises a surfactant, preferably polysorbate 20.

[0663] S16. The pharmaceutical composition as described in any one of S1 to S15 further comprises a sugar component, preferably trehalose.

[0664] S17. The pharmaceutical composition as described in any one of S1 to S16 further comprises an amino acid component, preferably arginine and / or lysine.

[0665] S18. The pharmaceutical composition as described in any one of S1 to S17 further comprises an antioxidant, preferably methionine.

[0666] Based on experimental studies, the following pharmaceutical compositions disclosed in T1 to T19 have been shown to be particularly stable:

[0667] T1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a buffer system, NaCl; a sugar component, a nonionic surfactant, and optionally an amino acid antioxidant, characterized in that the pH is 5.0-6.0.

[0668] T2. The pharmaceutical composition as described in T1, comprising 2-30 mg / mL IgG:TGFβR fusion protein, 2-30 mM buffer system, 20-70 mM NaCl; 100-200 mM sugar component, 0.1-1.1 mg / mL nonionic surfactant and optionally 1-20 mM amino acid antioxidant, characterized in that the pH is pH 5.0-6.0.

[0669] T3. The pharmaceutical composition as described in T1, comprising 5-15 mg / mL IgG:TGFβR fusion protein, 5-15 mM buffer system, 30-50 mM NaCl; 150-170 mM sugar component, 0.3-0.7 mg / mL nonionic surfactant and optionally 2-8 mM amino acid antioxidant, characterized in that the pH is 5.3-5.7.

[0670] T4. The pharmaceutical composition as described in T1, comprising about 10 mg / mL IgG:TGFβR fusion protein, about 10 mM buffer system, about 40 mM NaCl; about 159 mM sugar component, about 0.5 mg / mL nonionic surfactant and optionally about 5 mM amino acid antioxidant, characterized by a pH of about 5.5.

[0671] T5. The pharmaceutical composition as described in T1, comprising 25-100 mg / mL IgG:TGFβR fusion protein, 2-30 mM buffer system, 40-100 mM NaCl; 100-200 mM sugar component, 0.1-1.1 mg / mL nonionic surfactant and optionally 1-20 mM amino acid antioxidant, characterized in that the pH is pH 5.0-6.0.

[0672] T6. The pharmaceutical composition as described in T1, comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, 50-70 mM NaCl; 150-170 mM glycosides, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM amino acid antioxidant, characterized in that the pH is 5.3-5.7.

[0673] T7. The pharmaceutical composition as described in T1, comprising about 40 mg / mL IgG:TGFβR fusion protein, about 10 mM buffer system, about 60 mM NaCl; about 159 mM sugar component, about 0.5 mg / mL nonionic surfactant and optionally about 5 mM amino acid antioxidant, characterized by a pH of about 5.5.

[0674] T8. The pharmaceutical composition as described in any one of T1 to T7, wherein the IgG:TGFβR fusion protein has the same amino acid sequence as bintrafusp alfa, the buffer system is a histidine buffer system, the sugar component is trehalose, the nonionic surfactant is polysorbate 20, and the amino acid antioxidant is methionine.

[0675] T9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a buffer system, NaCl; a sugar component, an amino acid component, a nonionic surfactant, and optionally an amino acid antioxidant, characterized in that the pH is 5.4-6.4.

[0676] T10. The pharmaceutical composition as described in T9, comprising 25-100 mg / mL IgG:TGFβR fusion protein, 2-30 mM buffer system, 40-100 mM NaCl; 40-160 mM sugar component, 10-100 mM amino acid component, 0.1-1.1 mg / mL nonionic surfactant and optionally 1-20 mM amino acid antioxidant, characterized in that the pH is 5.4-6.4.

[0677] T11. The pharmaceutical composition as described in T9, comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, 50-70 mM NaCl; 90-110 mM glycosides, 40-60 mM amino acids, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM amino acid antioxidants, characterized in that the pH is 5.7-6.1.

[0678] T12. The pharmaceutical composition as described in T9, comprising about 40 mg / mL IgG:TGFβR fusion protein, about 10 mM buffer system, about 60 mM NaCl; about 100 mM sugar component, about 50 mM amino acid component, about 0.5 mg / mL nonionic surfactant and optionally about 5 mM amino acid antioxidant, characterized by a pH of about 5.9.

[0679] T13. The pharmaceutical composition as described in T9, comprising 25-100 mg / mL IgG:TGFβR fusion protein, 2-30 mM buffer system, 40-100 mM NaCl; 25-125 mM glycosides, 25-125 mM amino acid components, 0.1-1.1 mg / mL nonionic surfactant, and optionally 1-20 mM amino acid antioxidant, characterized in that the pH is 5.4-6.4.

[0680] T14. The pharmaceutical composition as described in T9, comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, 50-70 mM NaCl; 65-85 mM glycosides, 65-85 mM amino acids, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM amino acid antioxidants, characterized by a pH of 5.7-6.1.

[0681] T15. The pharmaceutical composition as described in T9, comprising about 40 mg / mL IgG:TGFβR fusion protein, about 10 mM buffer system, about 60 mM NaCl; about 75 mM sugar component, about 75 mM amino acid component, about 0.5 mg / mL nonionic surfactant and optionally about 5 mM amino acid antioxidant, characterized by a pH of about 5.9.

[0682] T16. The pharmaceutical composition as described in T9, comprising 35-120 mg / mL IgG:TGFβR fusion protein, a 2-30 mM buffer system, 50-150 mM NaCl; 10-100 mM glycosides, 10-100 mM amino acids, 0.1-1.1 mg / mL nonionic surfactant, and optionally 1-20 mM amino acid antioxidant, characterized by a pH of 5.4-6.4.

[0683] T17. The pharmaceutical composition as described in T9, comprising 45-55 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, 90-110 mM NaCl; 40-60 mM glycosides, 40-60 mM amino acids, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM amino acid antioxidants, characterized by a pH of 5.7-6.1.

[0684] T18. The pharmaceutical composition as described in T9, comprising about 50 mg / mL IgG:TGFβR fusion protein, about 10 mM buffer system, about 100 mM NaCl; about 50 mM sugar component, about 50 mM amino acid component, about 0.5 mg / mL nonionic surfactant and optionally about 5 mM amino acid antioxidant, characterized by a pH of about 5.9.

[0685] T19. The pharmaceutical composition as described in any one of T1 to T18, wherein the IgG:TGFβR fusion protein has the same amino acid sequence as bintrafusp alfa, the buffer system is a histidine buffer system, the sugar component is trehalose, the amino acid component is arginine, the nonionic surfactant is polysorbate 20, and the amino acid antioxidant is methionine.

[0686] In addition to the above, the following embodiments U1 to U21 are particularly preferred:

[0687] U1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an ionic tension modulator, and one or both of a disaccharide and an amino acid component.

[0688] U2. The pharmaceutical composition as described in U1, wherein the composition comprises a disaccharide.

[0689] U3. The pharmaceutical composition as described in U1 or U2, wherein the ion tension modifier is sodium chloride.

[0690] U4. The pharmaceutical composition of any one of U1 to U3, wherein the disaccharide is trehalose or sucrose.

[0691] U5. The pharmaceutical composition as described in any one of U1 to U4, wherein the disaccharide is trehalose.

[0692] U6. The pharmaceutical composition as described in any one of U1 to U5, wherein the amino acid component is arginine or lysine.

[0693] U7. The pharmaceutical composition as described in any one of U1 to U6, wherein the amino acid component is arginine.

[0694] U8. The pharmaceutical composition as described in any one of U1 to U7, wherein the ion tension modifier is sodium chloride, the disaccharide is trehalose, and the amino acid component is arginine.

[0695] U9. The pharmaceutical composition as described in any one of U1 to U8, further comprising a surfactant.

[0696] U10. A pharmaceutical composition as described in any one of U1 to U9, wherein the composition has a pH of 5.0 to 6.5.

[0697] U11. The pharmaceutical composition of any one of U1 to U10, wherein the IgG:TGFβR fusion protein is an anti-PD-1(IgG):TGFβR fusion protein or an anti-PD-L1(IgG):TGFβR fusion protein, preferably an anti-PD-1(IgG):TGFβR fusion protein.

[0698] U12. The pharmaceutical composition as described in U11, wherein the IgG:TGFβR fusion protein is an anti-PD-L1 (IgG):TGFβR fusion protein, and the anti-PD-L1 (IgG) is selected from the group consisting of: (1) anti-PD-L1 (IgG) comprising three heavy chain CDRs having the following amino acid sequences: SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3) and three light chain CDRs having the following amino acid sequences: SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3), (2) anti-PD-L1 (IgG) comprising three heavy chain CDRs having the following amino acid sequences: SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:3 (CDR3) and three light chain CDRs having the following amino acid sequences: SEQ ID NO:4 (CDR1), SEQ ID NO:24 (CDR3), and SEQ ID NO:25 (CDR3). NO:5 (CDR2) and SEQ ID NO:6 (CDR3), and (3) anti-PD-L1 (IgG) comprising three heavy chain CDRs having the following amino acid sequences: SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2) and SEQ ID NO:29 (CDR3) and three light chain CDRs having the following amino acid sequences: SEQ ID NO:30 (CDR1), SEQ ID NO:31 (CDR2) and SEQ ID NO:32 (CDR3).

[0699] U13. The pharmaceutical composition as described in U12, wherein the light chain sequence and heavy chain sequence of the anti-PD-L1 (IgG) correspond to (1) SEQ ID NO:7 and SEQ ID NO:16, (2) SEQ ID NO:15 and SEQ ID NO:14, or (3) SEQ ID NO:33 and SEQ ID NO:35, respectively.

[0700] U14. The pharmaceutical composition of any one of U1 to U13, wherein the TGFβR of the IgG:TGFβR fusion protein is the soluble extracellular domain of TGFβR2 or a fragment thereof capable of binding TGF-β.

[0701] U15. The pharmaceutical composition as described in U14, wherein TGFβR2 comprises or consists of sequences selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0702] U16. The pharmaceutical composition of any one of U1 to U15, wherein the light chain sequence and heavy chain sequence of the IgG:TGFβR fusion protein correspond to (1) SEQ ID NO:7 and SEQ ID NO:8, (2) SEQ ID NO:15 and SEQ ID NO:17, (3) SEQ ID NO:15 and SEQ ID NO:18, or (4) SEQ ID NO:33 and SEQ ID NO:34, respectively.

[0703] U17. The pharmaceutical composition of any one of U1 to U16, wherein the IgG:TGFβR fusion protein has the amino acid sequence bintrafusp alfa.

[0704] U18. A pharmaceutical composition as described in any one of U1 to U17, wherein the composition is characterized by a pH of 5.0 to 6.5 and comprises a buffer system, an ionic tension modifier, a disaccharide, a nonionic surfactant, an optional amino acid component, and a further optional antioxidant.

[0705] U19. The pharmaceutical composition as described in U18, wherein the buffer system is a histidine buffer system, the ion tension modifier is sodium chloride, the disaccharide is trehalose, the amino acid component is arginine, the antioxidant is methionine, and the nonionic surfactant is polysorbate 20.

[0706] U20. The pharmaceutical composition as described in U18 or U19, wherein the composition is selected from the group consisting of:

[0707] a. A pharmaceutical composition characterized by a pH of 5.3-5.7 and comprising 5-15 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, a 30-50 mM ion tonicity modifier, 150-170 mM disaccharide, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM antioxidant;

[0708] b. A pharmaceutical composition characterized by a pH of 5.3-5.7 and comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, a 50-70 mM ion tonicity modifier, 150-170 mM disaccharide, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM antioxidant;

[0709] c. A pharmaceutical composition characterized by a pH of 5.7-6.1 and comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, a 50-70 mM ion tonicity modifier; 90-110 mM disaccharide, a 40-60 mM amino acid component, a 0.3-0.7 mg / mL nonionic surfactant, and optionally a 2-8 mM antioxidant;

[0710] d. A pharmaceutical composition characterized by a pH of 5.7-6.1 and comprising 35-45 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, a 50-70 mM iontophoresis modifier; 65-85 mM disaccharide, 65-85 mM amino acid components, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM antioxidant; and

[0711] e. A pharmaceutical composition characterized by a pH of 5.7-6.1 and comprising 45-55 mg / mL IgG:TGFβR fusion protein, a 5-15 mM buffer system, a 90-110 mM ion tension modifier; 40-60 mM disaccharide, a 40-60 mM amino acid component, 0.3-0.7 mg / mL nonionic surfactant, and optionally 2-8 mM antioxidant.

[0712] U21. The pharmaceutical composition of any one of U1 to U20, wherein the ion tension modifier is a non-buffered ion tension modifier.

[0713] container

[0714] The present invention provides a container containing a pharmaceutical composition as defined herein. Preferably, the container includes a drug delivery device as defined herein, and preferably a plurality of drug delivery devices.

[0715] The present invention provides a method for manufacturing a container, the method comprising incorporating a pharmaceutical composition as defined herein into the container. Preferably, this is achieved by incorporating the pharmaceutical composition into one or more drug delivery devices, and then incorporating one or more pre-filled drug delivery devices into the container.

[0716] Drug delivery device

[0717] This invention provides a drug delivery device comprising a pharmaceutical composition as defined herein. Preferably, the drug delivery device includes a chamber in which the said, preferably liquid (most preferably aqueous) pharmaceutical composition resides. Preferably, the drug delivery device is sterile.

[0718] The drug delivery device can be a vial, ampoule, syringe, injection pen (e.g., substantially incorporated into a syringe), auto-injector, or intravenous infusion bag. Most preferably, the drug delivery device is a syringe. Preferably, the syringe is a glass syringe. Preferably, the syringe includes a needle.

[0719] This invention provides a method for manufacturing a drug delivery device, preferably as defined herein, the method comprising incorporating a drug composition as defined herein into the drug delivery device. This manufacturing typically involves loading the drug composition as defined herein into a syringe, preferably via a needle fixed thereto. The needle may thereafter be removed, replaced, or retained.

[0720] Multi-component reagent kit

[0721] The present invention provides a kit comprising a drug delivery device (in which no drug composition is contained), a drug composition as defined herein (optionally contained in a separate package or container), and optionally a set of instructions with instructions for administration of the drug composition (e.g., intravenous, subcutaneous). The user can then fill the drug delivery device with the drug composition (which may be provided in a vial or ampoule, etc.) prior to administration.

[0722] Manufacturing method

[0723] This invention provides a method for manufacturing a pharmaceutical composition as defined herein. The method preferably comprises mixing any relevant components required to form the composition as defined herein in any specific order deemed suitable. Those skilled in the art can refer to examples or techniques well known in the art to form pharmaceutical compositions (especially those for injection by syringe, and particularly those for intravenous injection). Different embodiments will preferably require different combinations of components mixed in potentially different amounts. Such combinations and amounts can be readily deduced by those skilled in the art from the foregoing disclosure relating to pharmaceutical compositions.

[0724] Preferably, the method includes mixing the relevant components together, preferably in a diluent (e.g., water), preferably such that all components are (substantially or completely) dissolved in the diluent.

[0725] The method may include first preparing a concentrated premix (or presolution) having some or all of the components, including the fusion protein, (optionally with some or all of the diluent), then diluting the premix (or presolution) with a diluent (preferably water) to provide a pharmaceutical composition, or subsequently adding the final component to provide a final pharmaceutical composition. Preferably, the premix is ​​prepared to have the pH required for the final formulation.

[0726] The method may alternatively or additionally include, firstly, preparing a premix (or presolution) having some or all of the components excluding the fusion protein (optionally with some or all of the diluents), then mixing the fusion protein itself (optionally with some diluents or pre-dissolved in some diluents) with the premix (or presolution) to provide a pharmaceutical composition, or subsequently adding the final components to it to provide a final pharmaceutical composition. Preferably, the premix contains all the components except the fusion protein and optionally also contains some diluents (which can be used to pre-dissolve the fusion protein), preferably such that the fusion protein is added to the mixture, which provides optimal stability of the fusion protein. Preferably, the premix is ​​prepared to have the pH required for the final formulation.

[0727] Preferably, the method includes forming a buffer system, preferably a buffer system comprising a buffering agent as defined herein. The buffer system may preferably have a pH as defined herein with respect to the buffer system itself. The buffer system is preferably formed in a premix before the addition of the fusion protein, although the buffer system may optionally be formed together with the fusion protein present. The buffer system can be formed by simply mixing a buffering agent (officially provided) with its acid / base conjugate (preferably in appropriate relative amounts to provide the desired pH – this can be determined theoretically or experimentally by a person skilled in the art). In the case of an acetate buffer system, this means mixing sodium acetate with acetic acid. Alternatively, the buffer system can be formed by adding a strong acid (e.g., HCl) to the buffering agent (e.g., sodium acetate) to form an acid / base conjugate (e.g., acetic acid) in situ (again preferably to provide the desired pH in appropriate relative amounts). Alternatively, the buffer system can be formed by adding a strong base (e.g., sodium hydroxide) to the acid / base conjugate (e.g., acetic acid) of the buffering agent (e.g., sodium acetate) to form the buffering agent in situ (again preferably to provide the desired pH in appropriate relative amounts). The pH of the premixture of the final composition can be reasonably adjusted by adding the required amount of a strong base or strong acid, or even by adding a certain amount of buffer or acid / base conjugate. The same approach can be used for other buffering systems, such as histidine buffering systems, which can be formed by combining free (neutral) histidine with histidine in the form of imidazole, or by adjusting the pH accordingly (e.g., using a base or acid as needed).

[0728] In some embodiments, the buffer and / or buffer system are pre-formed as a separate mixture, and the buffer system is transferred to the precursor of the composition (comprising some or all of the components other than the buffer and / or buffer system, preferably containing a fusion protein) via buffer exchange (e.g., using osmosis until the relevant concentration or wt. osmolar concentration is reached). If necessary, additional excipients may then be added to produce the final composition. The pH may be adjusted all at once or before all components are present.

[0729] Any, some, or all components may be pre-dissolved or pre-mixed with the diluent before being mixed with other components.

[0730] The final composition can be filtered, preferably to remove particulate matter. Filtration is preferably performed through a filter with a size of 1 μm or less, preferably 0.22 μm. Preferably, filtration is performed through a PES filter or a PVDF filter, with a 0.22 μm PES filter being more preferred.

[0731] It should be understood that the pharmaceutical compositions of the present invention may be provided in the form of a liquid (preferably aqueous) pharmaceutical composition and are preferably stored appropriately. However, the pharmaceutical compositions of the present invention may be provided in the form of a lyophilized pharmaceutical composition and are preferably stored appropriately. Such lyophilized pharmaceutical compositions may be formed by lyophilizing a liquid (e.g., aqueous) pharmaceutical composition as defined herein. However, most preferably, any such lyophilized pharmaceutical composition will be reconstituted before use, administration, or even (possibly short-term) storage to provide a liquid or aqueous pharmaceutical composition as defined herein. Such reconstitution preferably involves dissolving the lyophilized composition in water (for injection), preferably providing a solution having the desired concentration of the fusion protein.

[0732] Treatment

[0733] This invention provides a method for treating a disease or medical condition by administering a pharmaceutical composition as defined herein. The method preferably includes administering a therapeutically effective amount of the pharmaceutical composition to a subject in need. Administration may preferably include parenteral administration, which preferably includes any form of administration other than enteral and local administration, typically by injection, and preferably includes, but is not limited to, intravenous, intravitreal, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. The method preferably includes subcutaneous or intravenous delivery of a therapeutically effective amount of the pharmaceutical composition. The method preferably includes intravenous administration of a therapeutically effective amount of the pharmaceutical composition, preferably by intravenous infusion, and preferably in conjunction with other intravenous infusions (e.g., saline).

[0734] When the pharmaceutical composition is a lyophilized pharmaceutical composition, it is preferable to reconstitute the composition in water for injection or a suitable intravenous fluid before administration, preferably providing an aqueous pharmaceutical composition as defined herein. The aqueous pharmaceutical composition is then preferably administered as defined herein, preferably by intravenous injection, preferably together with other intravenous fluids (e.g., saline).

[0735] This invention provides pharmaceutical compositions as defined herein for treating diseases or medical conditions in patients requiring such treatment. This invention also provides the use of the pharmaceutical compositions as defined herein in the preparation of medicaments for treating diseases or medical conditions.

[0736] This invention provides methods for treating diseases or medical conditions, pharmaceutical compositions for treating diseases or medical conditions, and the use of pharmaceutical compositions in the preparation of medicaments for treating diseases or medical conditions as defined herein, wherein said diseases or medical conditions are PD-L1- and / or TGFβ-related diseases, preferably PD-L1- and / or TGFβ-related proliferative diseases, more preferably PD-L1- and / or TGFβ-related cancers. In these aspects of the invention, the anti-PD-L1 antigen-binding portion of the anti-LD-L1(IgG):TGFβR fusion protein can bind to tumor-carrying PD-L1, thereby disrupting the PD-1 / PD-L1 pathway, while simultaneously localizing the TGFβ receptor portion of the fusion protein in the tumor microenvironment, which can most effectively target and / or isolate autocrine or paracrine TGFβ. In this way, multiple mechanisms of action are disrupted, thereby blocking cancer cell growth and / or survival pathways.

[0737] This invention provides methods for treating diseases or medical conditions, pharmaceutical compositions for treating diseases or medical conditions, and the use of pharmaceutical compositions in the preparation of medicaments for treating diseases or medical conditions as defined herein, wherein said diseases or medical conditions are proliferative diseases or conditions. Proliferative diseases or conditions are preferably cancer. Cancer is preferably manifested in one or more solid tumors. Preferably, the cancer is selected from carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specific examples of these cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (intestinal) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, biliary tract cancer, and head and neck cancer. The disease or medical condition discussed may preferably be selected from any of those disclosed in WO2015118175, WO2018029367, WO2018208720, PCT / US18 / 12604, PCT / US19 / 47734, PCT / US19 / 40129, PCT / US19 / 36725, PCT / US19 / 732271, PCT / US19 / 38600 and PCT / EP2019 / 061558.

[0738] This invention provides methods for treating diseases or medical conditions, pharmaceutical compositions for treating diseases or medical conditions, and the use of pharmaceutical compositions in the preparation of medicaments for treating diseases or medical conditions as defined herein, wherein the diseases or medical conditions are preferably as defined herein, and wherein the treatment or therapy involves combination therapy, whereby the pharmaceutical composition is administered in combination with one or more other pharmaceutical or bioactive substances; wherein the combination therapy involves the simultaneous, sequential, or separate administration of the components of the treatment. In some embodiments, additional pharmaceutical or bioactive substances may be present in any pharmaceutical composition as defined herein.

[0739] All references cited in this article are incorporated into the disclosure of this invention through citation.

[0740] Example

[0741] Anti-PD-L1 bioactivity assay

[0742] The bioactivity of the anti-PD-L1(IgG):TGFβRII fusion protein was assessed using a cell-based assay that measures its ability to bind to the human PD-L1 receptor overexpressed on the recombinant HEK-293(hPD-L1) cell line. For this purpose, the anti-PD-L1(IgG):TGFβRII fusion protein was vortexed with a protein A-coated 96-well plate for 30 min at room temperature. Then, 50,000 HEK-293(hPD-L1) cells were added to each well of the anti-PD-L1(IgG):TGFβRII-coated plate, and the cells were bound to the anti-PD-L1(IgG):TGFβRII for 1 h at 37°C and 5% CO2. Unbound cells were washed away, and the presence of bound cells in each well was confirmed by a one-step ATPlite assay (PerkinElmer). The counts per second were then plotted against logarithmically transformed anti-PD-L1(IgG):TGFβRII concentrations and fitted using a 4PL (sigmoid dose-response curve). For each dataset, the concentration at which anti-PD-L1(IgG):TGFβRII could exert its maximum possible PDL-1 binding capacity (EC50) was calculated. Bioactivity of the samples is expressed as a percentage of activity relative to a reference material, i.e., a percentage of potency (i.e., EC50 of an equivalent dose of standard compared to an unknown formulation). Potency is the average of three independent assays.

[0743] TGFβRII Bioactivity Assay

[0744] The TGFβ binding activity of the anti-PD-L1(IgG):TGFβRII fusion protein was assessed using the reporter cell line 4T1 pSmad sLuc, which was stably transfected with a luciferase reporter gene controlled by a SMAD-binding element. Upon binding of TGFβ to the TGFβ receptor on the cell surface of the cell line, a signaling cascade is triggered, leading to the formation of the SMAD complex. The SMAD complex then translocates to the nucleus, where it binds to the SMAD-binding element and induces transcription of the luciferase gene.

[0745] For each bioassay, 50,000 4T1 cells / well were loaded into 96-well plates and allowed to adhere to the plates for 4 hours at 37°C and 5% CO2. The plates were then incubated overnight at 37°C and 5% CO2 with different concentrations of anti-PD-L1(IgG):TGFβRII fusion protein and a fixed amount of human TGFβ to generate dose-response curves. Luciferase expression was subsequently assessed using a fluorescein-based detection dye. The emitted light was negatively correlated with the TGFβ binding activity of the anti-PD-L1(IgG):TGFβRII fusion protein, as the anti-PD-L1(IgG):TGFβRII fusion protein captures and neutralizes soluble TGFβ.

[0746] For each dataset, the concentration at which the anti-PD-L1(IgG):TGFβRII can exert 50% of its maximum possible TGFβ binding capacity (EC50) was calculated. The bioactivity of the sample is expressed as a percentage of activity relative to a reference material, i.e., a percentage of potency ratio (i.e., EC50 of an equivalent dose of standard compared to an unknown formulation). Potency is the average of three independent assays.

[0747] Stress testing solution

[0748] The following stress testing scheme was used in the following embodiments, although variations thereof will be apparent from the context.

[0749] thermal stability

[0750] The thermal stability of the formulation is examined during a storage period at 40±2℃ (75% RH) for a period of time (e.g., 8, 9, or 13 weeks). Samples are analyzed not only at time = 0 and time = the full period (e.g., 8 weeks), but also at intermediate intervals (e.g., time = 2 weeks, time = 4 weeks). Sometimes, spare samples are placed at 25±5℃ for a similar duration to allow analysis of samples that degrade too rapidly or produce abnormal results at 40±2℃. The following analyses are typically performed on samples for thermal stability:

[0751] ● Protein content detection using optical density

[0752] ● Aggregation Index: Calculated using optical density to track the aggregation and formation of HMW impurities.

[0753] ● HMW content detection via SE-UPLC (to track the generation of HMW impurities and thus monitor their aggregation)

[0754] ● Turbidity is determined by turbidimetric methods (which are also related to solubility and aggregate formation).

[0755] ● Visual inspection may reveal the presence of particles and / or changes in color intensity.

[0756] ● Detection of charged isotypes (monitoring charge variants) by isoelectric capillary electrophoresis

[0757] ●pH

[0758] ● LMW content was detected using a bioanalyzer or CGE-NR (fragmentation tracking).

[0759] ●Detection of sub-visible particles using the light-shielding method

[0760] ● Fluorescence detection (on selected samples) to monitor changes in molecular tertiary structure.

[0761] Light pressure

[0762] The photosensitivity of the IgG:TGFβR2 fusion protein in each formulation was also investigated. The formulations were typically exposed to 765 W / m for 7 hours. 2 Under light intensity of [insert intensity here], this meets the requirements of ICH Q1B guidelines. Formulations subjected to light stress are typically analyzed using the following techniques:

[0763] ● Protein content detection using optical density

[0764] ● Aggregation Index: Calculated by OD, it measures the degree of aggregation caused by light pressure.

[0765] ● Turbidity is measured by turbidimetric method.

[0766] ●Visual inspection: Check for visible particles caused by aggregation.

[0767] ●SE-UPLC: Quantitative analysis of HMW impurities generated by aggregation.

[0768] ● Detection of charged isotypes (monitoring charge variants) by isoelectric capillary electrophoresis

[0769] ●Fluorescence (selected sample): Observe any tertiary structural changes that may be caused by the oxidation of aromatic amino acid side chains.

[0770] Oscillation pressure

[0771] Mechanical (oscillatory) stress is generally associated with aggregate formation due to interactions between self-association and hydrophobic regions of proteins in solution. Anti-oscillatory stress was studied in various formulations of the IgG:TGFβR2 fusion protein, typically at 200 mg / vial and 600 mg / vial, stirred at 200 rpm (or 300 rpm) in both upright and inclined positions for 24 hours at room temperature. The oscillatory stress formulations were generally analyzed as follows:

[0772] ● Protein content detection using optical density

[0773] ● Aggregation Index: Calculated using optical density to track the aggregation and formation of HMW impurities.

[0774] ● Turbidity is determined by turbidimetric methods (which are also related to solubility and aggregate formation).

[0775] ● Visually inspect for visible particles and / or discoloration.

[0776] ● HMW content detection via SE-UPLC (to track the generation of HMW impurities and thus monitor polymerization)

[0777] ● LMW content was detected using a bioanalyzer or CGE-NR (fragmentation tracking).

[0778] ●Detection of sub-visible particles using the light-shielding method

[0779] ●pH

[0780] Freezing / thawing pressure

[0781] When protein formulations are frozen, interfaces form as microregions in the solution begin to solidify. Within these microenvironments, polarities change as different components of the formulation buffer are excluded from or incorporated into the solidifying liquid matrix. This results in protein precipitation because hydrophilic / hydrophobic interactions are imposed on the molecules within these changing microenvironments. To determine the effectiveness of various excipients and conditions, formulations of the IgG:TGFβR2 fusion protein are exposed to three freeze-thaw cycles (each cycle from -25°C to RT), typically at 200 mg / vial or 600 mg / vial samples. The samples are then typically examined by the following analyses to determine their resistance to freeze-thaw precipitation / aggregation / degradation:

[0782] ● Protein content detection using optical density

[0783] ● Aggregation Index: Calculated using optical density to track the aggregation and formation of HMW impurities.

[0784] ● Detection of charged isotypes (monitoring charge variants) by isoelectric capillary electrophoresis

[0785] ● Turbidity is determined by turbidimetric methods (which are also related to solubility and aggregate formation).

[0786] ● Visually inspect for visible particles.

[0787] ● HMW content detection via SE-UPLC (to track the generation of HMW impurities and thus monitor polymerization)

[0788] ● LMW content was detected using a bioanalyzer or CGE-NR (fragmentation tracking).

[0789] ●Subvisible particles

[0790] ●pH

[0791] Results Analysis and Data Processing Scheme

[0792] Several Design of Effect (DoEs) were set up using the Response Surface Model option in Design-Expert software, selecting a D-optimal design with three numerical factors (buffer strength, pH, surfactant strength) and one categorical factor (excipient type). To determine whether there was a positive response to a given pressure, the data were statistically evaluated using a linear response surface model via analysis of variance. The relationships between all variables in the DoEs were explored using Design Expert software, and the results are illustrated with 3D surface plots to demonstrate the important influencing factors on the response to a given pressure. This approach establishes a statistically significant relationship between given factors and the response to pressure.

[0793] Formulation preparation scheme

[0794] The bintrafusp alfa drug substance (an example of an IgG:TGFβR2 fusion protein) can be manufactured as described in WO2015118175, the examples of which illustrate its production. The complete heavy chain of IgG linked to the TGFβR2 extracellular domain via a linker is shown in SEQ ID NO:3. The TGFβR2 extracellular domain corresponds to SEQ ID NO:10, while the linker corresponds to SEQ ID NO:11. The light chain of bintrafusp alfa corresponds to SEQ ID NO:1 of WO2015118175.

[0795] Figure 1A Half of the bintrafusp alfa fusion protein was shown, which contains a light chain (with a specified V) that is linked to the extracellular domain (ECD) of TGFβ receptor II via a designated linker. L and C L (region) and heavy chain (with specified V)H C H 1. C H 2 and C H (District 3).

[0796] Figure 1B The sequence of half of the bintrafusp alfa fusion protein is also shown, which contains a light chain (with designated VL and CL regions) and a heavy chain (with designated VH, CH1, CH2, and CH3 regions) that are linked to the extracellular domain (ECD) of TGFβ receptor II via designated linkers (see above regarding...). Figure 1A and 1B (Further information).

[0797] Purified bintrafusp alfa was stored in a pre-prepared state at -70°C and thawed before use.

[0798] In one method, an aqueous formulation is prepared by mixing a pre-prepared solution containing excipients (at a specified pH, where applicable) with a suitable weight of bintrafusp alfa to provide the desired formulation based on the concentrations of bintrafusp alfa and excipients. In some cases, a final pH adjustment is performed to ensure the pH meets expectations. In other methods, an aqueous formulation can be produced by dissolving or mixing bintrafusp alfa in water, optionally containing some excipients, and then adding more excipients (optionally pre-dissolved in water) to provide the final desired formulation—again, a final pH adjustment may be performed.

[0799] In some methods, a stock solution of bintrafusp alfa is prepared (e.g., by the methods described above) and may be stored at a reduced temperature before use / processing. The stock solution can then be processed to provide the desired formulation, for example by performing buffer exchange, concentration, dilution, etc.

[0800] In the following embodiments, how to produce the relevant formulations is self-evident to those skilled in the art.

[0801] Example 1 – Lyophilized and Liquid Formulations of IgG:TGFβR2 Fusion Protein

[0802] General Overview

[0803] Preparing stable formulations for IgG:TGFβR2 fusion proteins is particularly challenging, partly due to the fact that the two fused entities, the IgG moiety and the TGFβR2 moiety, are not well-suited in terms of pI and colloidal stability—the pI of the TGFβR2 region is completely different from that of the IgG moiety, but it is generally considered important to use a pH that is at least 1-2 pH units away from the pI of each major protein moiety.

[0804] List of test formulations

[0805] Preliminary screening was conducted to establish feasible and optimal pH values, focusing on colloidal and conformational stability. Formulations selected for this preliminary screening are shown in Table 1A.

[0806] Table 1A – Formulations for initial pH screening

[0807]

[0808]

[0809] *The buffer used in this embodiment is a composite buffer system containing 10 mM sodium phosphate, 10 mM TRIS, and 10 mM citrate.

[0810] Further experiments were conducted to determine the effect of sodium chloride on solubility and dialysis recovery. These formulations are shown in Table 1B.

[0811] Table 1B shows formulations used for initial sodium chloride and solubility screening.

[0812]

[0813]

[0814] Then, 18 pH 5.5 formulations containing different concentrations of sodium chloride and trehalose (wt% of trehalose relative to wt% of trehalose dihydrate) were further screened for lyophilizability and osmolality to evaluate lyophilization performance. APIs were excluded from these formulations because they had little effect on the observed factor (appearance of the lyophilized product) and their effect on osmolality was negligible. Such placebo formulations can therefore be considered the worst-case scenario regarding the appearance of the lyophilized product. These formulations are shown in Table 1C.

[0815] Table 1C – Formulations of sodium chloride and lyophilization protectants used for screening lyophilizability and weight permeability molar concentration.

[0816] preparation pH NaCl Trehalose 1C.1 5.5 160mM 8wt% 1C.2 5.5 80mM 8wt% 1C.3 5.5 40mM 8wt% 1C.4 5.5 20mM 8wt% 1C.5 5.5 10mM 8wt% 1C.6 5.5 0mM 8wt% 1C.7 5.5 160mM 4wt% 1C.8 5.5 80mM 4wt% 1C.9 5.5 40mM 4wt% 1C.10 5.5 20mM 4wt% 1C.11 5.5 10mM 4wt% 1C.12 5.5 0mM 4wt% 1C.13 5.5 160mM 2wt% 1C.14 5.5 80mM 2wt% 1C.15 5.5 40mM 2wt% 1C.16 5.5 20mM 2wt% 1C.17 5.5 10mM 2wt% 1C.18 5.5 0mM 2wt%

[0817] Six essentially isotonic pH 5.5 formulations containing finely tuned concentrations of sodium chloride and trehalose were then further screened based on lyophilizability and gravimetric osmolality. These formulations are shown in Table 1D. Similarly, APIs were excluded for the same reasons, according to Table 1C.

[0818] Table 1D – Other formulations of sodium chloride and lyophilization protectants used for screening lyophilizability and weight permeation molar concentration.

[0819]

[0820] Two essentially isotonic pH 5.5 formulations were then further screened, containing 10 mg / mL bintrafuspalfa and finely tuned concentrations of sodium chloride and trehalose. These formulations are shown in Table 1E.

[0821] Table 1E – Formulations used for rapid stability screening

[0822]

[0823] Formulation 1A - Results and Analysis

[0824] Nanoscale DSC measurements were performed on formulations 1A.1–1A.8 (see Table 1A above) to measure the conformational stability of these formulations, and the results are presented in Table 1F below.

[0825] Table 1F-DSC results show T m 1 and T m 2. Protein unfolding events at temperature peaks

[0826]

[0827] The above T m Measurements show that, from a conformational stability perspective, the optimal pH is between 5.5 and 9. Furthermore, also from a conformational stability perspective, the addition of NaCl is not harmful, but it does not improve conformational stability itself. At pH 4 and 5, without any NaCl, T... m A slightly lower value indicates reduced conformational stability.

[0828] Formulation 1B - Results and Analysis

[0829] The solubility of the formulations listed in Table 1B above (formulations 1B.1-1B.18) was determined, with the percentage protein recovery measured after reconstitution / rebuffering to determine the colloidal stability of these formulations. The results are shown in Table 1G below.

[0830] Table 1G – Solubility Determination: Results show protein recovery after reconstitution / rebuffering.

[0831]

[0832]

[0833] Overall, pH 5.5 showed better solubility profiles than pH 7 at concentrations of 1–10 mg / mL. However, NaCl had a significant impact on protein recovery—especially at higher pH values.

[0834] 1C Formulation - Results and Analysis

[0835] The formulations in Table 1C (formulations 1C.1 to 1C.18) all have a pH of 5.5 and contain 10 mg / mL bintrafuspalfa, and are tested under lyophilized conditions (by standard freeze-thaw cycles), and the weight permeation molar concentration when composed of 10 mg / mL bintrafuspalfa is tested (using the freezing point depression method commonly used in the pharmaceutical industry).

[0836] The results are shown in Table 1H below.

[0837] Table 1H – Determination of freeze-dryability and gravimetric osmolality

[0838]

[0839]

[0840] - This is the weight permeation molar concentration of the formulation before the addition of bintrafusp alfa.

[0841] - Appearance is rated from 1 to 5 (from best to worst), where: 1 = completely intact freeze-dried product; 2 - slight shrinkage of bottom diameter (more than Tg'); 3 - more obvious shrinkage of bottom diameter (more than Tg'); 4 - bottom collapse (initial drying may not be complete); 5 - complete collapse.

[0842] Figure 2 The diagram shows the relationship between Tg' (expressed in °C) and NaCl concentration for formulations containing 8% trehalose (rhomboid), 4% trehalose (square), and 2% trehalose (triangle), determined by nanoscale differential scanning calorimetry. From a practical standpoint during freeze-drying, a Tg' value below -40 °C is undesirable.

[0843] Data indicate that a promising window exists around 40 mM NaCl from the perspective of balancing lyophilization activity and weight osmotic molar concentration (i.e., substantially isotonic formulation). Furthermore, from a lyophilization perspective, a trehalose concentration between 4% and 6% appears desirable. More generally, a molar ratio of tension-modifying lyophilization protectant to ion tension modifier of 10:1 to 1:2, more preferably 5:1 to 2:1, appears particularly suitable in terms of lyophilization properties.

[0844] 1D Formulation - Results and Analysis

[0845] Following experiments with formulations 1A-1C, a formulation window offering a favorable balance in terms of isotonicity, solubility, and lyophilization was determined. Formulations in Table 1D (Formulations 1D.1-1D.6), all with a pH of 5.5 and containing 10 mg / mL bintrafusp alfa, were tested for lyophilization and gravimetric osmolality in the same manner as formulation 1C, and the results are shown in Table 1I below.

[0846] Table 1I – Further determinations of lyophilizability and gravimetric osmolality (fine-tuning)

[0847]

[0848]

[0849] - This is the weight permeation molar concentration of the formulation before the addition of bintrafusp alfa.

[0850] - Appearance is rated from 1 to 5 (from best to worst), where: 1 = completely intact freeze-dried product; 2 - slight shrinkage of bottom diameter (more than Tg'); 3 - more obvious shrinkage of bottom diameter (more than Tg'); 4 - bottom collapse (initial drying may not be complete); 5 - complete collapse.

[0851] The results showed that formulations containing approximately 40 mM NaCl or 20-50 mM NaCl were optimal for 10 mg / mL bintrafuspalfa formulations, as lyophilization was compromised at 60 mM NaCl, while bintrafuspalfa began to precipitate at approximately 20 mM NaCl, as observed during sample dilution for analytical purposes. Furthermore, sugars, such as trehalose (sucrose is also an effective alternative), at around 6% (w / v) (≥4.4% (w / v) being preferred), exhibited different modes of action and could be used to formulate 10 mg / mL bintrafuspalfa into lyophilized dosage forms.

[0852] 1E Formulation - Results and Analysis

[0853] Based on the experiments with formulations 1A-1D, a formulation window providing a favorable balance between isotonicity and lyophilization was established. Further investigation was conducted to determine whether this window also provides sufficient stability for the protein. To this end, short-term (3-week) stability tests (at 5°C, 25°C, and 40°C) were performed on the two formulations in Table 1E. The results showed that although both formulations (lyophilized and liquid) passed the stability tests, the formulation with 75 mM NaCl and 4% trehalose (pH 5.5) was not sufficiently lyophilized for lyophilization. The liquid formulation of the drug (10 mg / mL bintrafusp alfa, 10 mM L-histidine, 5 mM methionine, 40 mM NaCl, 6% (w / v) trehalose, 0.05% (w / v) polysorbate 20, pH 5.5) indeed showed good stability (no significant changes in quality properties at 5°C for 3 months; although a slight increase in particle size was observed at 6 months, other quality properties remained within the expected range).

[0854] However, these early stability tests indicated that even when cooled to 5°C, liquid formulations of bintrafusp alfa may not exhibit long-term stability, thus suggesting that lyophilized formulations would provide a solution to this problem. However, it is known that using increased amounts of NaCl to dissolve high concentrations of bintrafusp alfa can adversely affect lyophilization, therefore, many issues remain regarding allowing for formulations with higher drug concentrations.

[0855] In other words, these studies suggest that lyophilized formulations offer a potential solution for formulations that exhibit certain challenges in solution-phase storage stability.

[0856] Conclusion of this embodiment

[0857] If the pH and / or NaCl concentration are maintained within an appropriate window for a given concentration of bintrafusp alfa, other formulation parameters, such as buffer systems, surfactants, and other excipients, can obviously be significantly varied without adversely affecting basic stability, solubility, lyophilizability, and osmotic molar concentration. For 10 mg / mL bintrafusp alfa, the following formulations have been confirmed as particularly suitable:

[0858]

[0859] Surprisingly, the optimal pH (pH 5.5) was within one pH unit of the pI of the TGFβR2 fraction. However, a wider pH range, such as between pH 5 and 9, appears to be acceptable, especially when sodium chloride is added.

[0860] Sodium chloride promotes the dissolution of drug substances, but too much NaCl will impair lyophilization.

[0861] However, at this stage, lyophilized formulations are considered the most likely to succeed. Indeed, lyophilized formulations offer a potential solution for formulations exhibiting certain solution-phase storage stability challenges. However, if sodium chloride is chosen as the provider of ionic strength, the maximum drug concentration of the lyophilized formulation may be limited, as higher drug concentrations require more NaCl to promote solubility and stability, but high salt concentrations can impair lyophilizability.

[0862] Example 2 – High-concentration liquid formulation of IgG:TGFβR2 fusion protein

[0863] General Overview

[0864] Although Example 1 provides a feasible pharmaceutical composition, it was desirable to increase the concentration of the IgG:TGFβR2 fusion protein, whether for use in lyophilized or liquid compositions, and to investigate whether these could withstand longer storage periods. Therefore, further formulation studies were conducted.

[0865] List of test formulations

[0866] Stability results indicated that the candidate formulation from Example 1 (2A.1) was also stable in liquid form, and therefore this formulation was used as the reference sample in Example 2. Four additional formulations (2A.2–2A.5) were then screened to evaluate the feasibility of upconcentrating bintrafusp alfa. These formulations are shown in Table 2A.

[0867] Table 2A – Concentrated Formulations

[0868]

[0869]

[0870] Results and Analysis

[0871] Table 2A lists formulations that underwent long-term stability studies (up to 12 months at 5°C), accelerated stability studies (up to 6 months at 25°C), and pressure stability studies (up to 3 months at 40°C). Table 2B shows the analytical results related to the long-term stability study (at 5°C) of formulation 2A.2 with a drug concentration of 20 mg / mL.

[0872] Table 2B – Results of long-term stability study of formulation 2A.2

[0873]

[0874]

[0875] Table 2C shows the analytical results related to the long-term stability study (at 5°C) of formulation 2A.3 with a drug concentration of 35 mg / mL.

[0876] Table 2C – Results of long-term stability study of formulation 2A.3

[0877]

[0878]

[0879] Table 2D shows the analytical results related to the long-term stability study (at 5°C) of formulation 2A.4 at a drug concentration of 40 mg / mL.

[0880] Table 2D – Results of the long-term stability study of formulation 2A.4

[0881]

[0882]

[0883] Table 2E shows the analytical results related to the long-term stability study (at 5°C) and the accelerated stability study (at 25°C), using formulation 2A.5 with a drug concentration of 40 mg / mL, but with the sodium chloride concentration increased to 60 mM.

[0884] Table 2E – Results of long-term and accelerated stability studies of formulation 2A.5

[0885]

[0886]

[0887]

[0888] Table 2F shows the protein stability of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C), measured by optical density. No particular decrease in protein concentration was observed in any of the analytical batches during these stability studies.

[0889] Table 2F – Protein concentration measured by optical density at different time points after storage at 5°C, 25°C, and 40°C in formulations 2A.2-2A.5

[0890]

[0891] Table 2G shows the percentage of high molecular weight substances (%HMW) in all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C), as measured by SE-UPLC. Higher concentrations result in higher %HMW, except for 2A.5 (60 mM NaCl), which appears to be more stable than the corresponding formulation with 40 mM NaCl (also containing 40 mg / mL bintrafusp alfa). This suggests that higher API concentrations require more NaCl to mitigate aggregation.

[0892] Table 2G – Formulation 2A.2-2A.5 %HMW at different time points after storage at 5°C, 25°C and 40°C

[0893]

[0894] Table 2H shows the percentage of low molecular weight substances (%LMW) in all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C), as measured by CE-SDS. Under all stability conditions, 2A.5 had a lower %LMW, which may indicate that NaCl can mitigate fragmentation in samples with higher drug concentrations.

[0895] Table 2H – Formulation 2A.2-2A.5 % LMW at different time points after storage at 5°C, 25°C and 40°C

[0896]

[0897]

[0898] Table 2I shows the % oxidation levels of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5 °C), accelerated stability testing (25 °C), and pressure stability testing (40 °C). Similar oxidation levels (%) were observed for all batches at 5 °C ± 3 °C and 25 °C ± 2 °C (considering the IRS oxidation levels reported as comparisons in each segment), while an increase was observed at 40 °C ± 2 °C. The high values ​​for 5 °C and 25 °C 2–6 M may be misleading due to the variability of the analytical sessions.

[0899] Table 2I – Formulation 2A.2-2A.5 % Oxidation at Different Time Points After Storage at 5℃, 25℃ and 40℃

[0900]

[0901]

[0902] Table 2J shows the percentage of deamination (LC) in all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C), as measured by IEX-HPLC. At 5°C ± 3°C and 25°C ± 2°C, all tested samples remained stable over time, with only a slight increase at the last time point for 2A.5. At 40°C ± 2°C, %LC increased over time. The deamination levels were comparable for all samples and were clearly independent of the API concentration in the formulation.

[0903] Table 2J – Formulations 2A.2-2A.5: % deamidation (LC) at different time points after storage at 5°C, 25°C, and 40°C.

[0904]

[0905] Table 2K shows the % purity of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C), as measured by CE-SDS RED. A slight decrease in purity was observed at 25°C ± 2°C, with a more pronounced decrease at 40°C ± 2°C. However, the trends for all formulations were comparable and appeared to be independent of concentration.

[0906] Table 2K – Formulation 2A.2-2A.5 % purity at different time points after storage at 5℃, 25℃ and 40℃

[0907]

[0908] Table 2L shows the % principal clipping (PPC) of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C). Under all conditions, the increase in PPC at all time points was similar for all formulations and therefore appears to be independent of concentration.

[0909] Table 2L – Formulation 2A.2-2A.5 % Main clipping at different time points after storage at 5℃, 25℃ and 40℃

[0910]

[0911] Table 2M shows the percentage of anti-PD-L1 bioactivity of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C). No significant changes in the bioactivity of the anti-PD-L1 fraction were observed over time under all stability conditions.

[0912] Table 2M – Formulation 2A.2-2A.5: % anti-PD-L1 bioactivity at different time points after storage at 5℃, 25℃, and 40℃.

[0913]

[0914]

[0915] Table 2N shows the % TGF-β bioactivity of all four concentrated formulations (2A.2–2A.5) in Table 2A at different time intervals (from time = 0 to 12 months) under long-term stability testing (5°C), accelerated stability testing (25°C), and pressure stability testing (40°C). A significant decrease in the bioactivity of the TGF-β fraction over time was observed at 40°C ± 2°C, with similar degrees of decrease across all formulations.

[0916] Table 2N – Formulations 2A.2-2A.5 % TGF-β bioactivity at different time points after storage at 5℃, 25℃ and 40℃

[0917]

[0918]

[0919] in conclusion

[0920] Table 2O summarizes the trends observed in the above stability studies.

[0921] Table 2O – Analytical protocols and general observations for testing formulations in Table 2A

[0922]

[0923]

[0924] Again, assuming that the pH and / or NaCl concentration are maintained within an appropriate window for a given concentration of bintrafusp alfa, other formulation parameters, such as buffer systems, surfactants, and other excipients, can be significantly varied without adversely affecting basic stability, solubility, lyophilizability, and osmotic molar concentration. For 40 mg / mL bintrafusp alfa, the following formulations have been identified as particularly suitable:

[0925]

[0926] The main differences from the lead candidate identified in Example 1 are a higher concentration of bintrafusp alfa, at 40 mg / mL instead of 10 mg / mL, and an increased amount of sodium chloride, from 40 mM to 60 mM. Furthermore, these studies indicate that the liquid formulations of Example 1 and this example are indeed feasible, and lyophilization / reconstitution may not be strictly necessary if the storage of the liquid formulation is considered more practical.

[0927] Further research also shows that, for a given concentration of bintrafusp alfa, the optimal amount of NaCl is as follows:

[0928] ●10–20 mg / mL API is stable with 40 mM NaCl

[0929] ●40 mg / mL, 60 mM NaCl is generally preferred.

[0930] ●60 mg / mL contains trehalose; 100 mM NaCl is generally preferred.

[0931] ●60mg / mL trehalose-free, generally 150mM NaCl is preferred.

[0932] These studies also demonstrate that liquid formulations with high concentrations of IgG:TGFβR2 are indeed feasible, and may even be superior to lyophilized formulations, especially when using higher concentrations of sodium chloride.

[0933] Example 3A – Further Screening Study – Step 1A (pH, Ionic Strength, and Protein Concentration Screening)

[0934] General Overview

[0935] Further formulation studies were conducted to evaluate changes in pH (pH 5.0–7.5), ionic strength (40–150 mM ionic salts, such as sodium chloride), and protein concentration (20, 40, and 60 mg / mL) in a 10 mM histidine buffer system, which served as an exemplary buffer system.

[0936] List of test formulations

[0937] Twenty formulations were initially screened to determine feasible and optimal pH, feasible and optimal ionic strength, and feasible and optimal protein concentration. The formulations screened in this initial screening are shown in Table 3A.

[0938] Table 3A – Formulations used for initial pH, ionic strength, and protein concentration screening

[0939]

[0940]

[0941] Results and Analysis

[0942] The formulations listed in Table 3A were evaluated in the DoE study. Specifically, their unfolding temperatures were examined. Furthermore, they were exposed to thermal stress (40°C, 4 weeks) and light stress (765 W / m²). 2 The samples were subjected to pressure for 7 hours and analyzed using various analytical techniques.

[0943] For samples subjected to thermal stress, important models can be constructed by referring to the following analysis, although other additional analytical techniques were also used during the preliminary analysis:

[0944] ●HMW, via SE-UPLC

[0945] ● Overview of similar types, via ICE3

[0946] ●Purity / LMW, via CGE-SDS (Red / NoRed)

[0947] ●Oxidized form, via RP-UPLC

[0948] ●Deamide form, as determined by IEX-HPLC

[0949] ●Defolding temperature, via nano-DSC

[0950] For samples subjected to light stress, important models can be constructed by referring to the following analysis, although other additional analytical techniques were also used again during the preliminary analysis:

[0951] ●HMW, via SE-UPLC

[0952] ●Oxidized form, via RP-UPLC

[0953] ● Overview of similar types, via iCE3

[0954] Defolding temperature

[0955] Figure 3 and Figure 4Together, this suggests that, from the perspective of unfolding temperature, a pH range of 5.7–6.2 may be optimal, and that higher protein concentrations require higher ionic strength to provide conformational stability.

[0956] Table 3B shows the unfolding temperatures of the formulations in Table 3A as measured by nano-DSC.

[0957] Table 3B–Table 3A formulations unfolding temperatures (T) m 1-T m 4)

[0958]

[0959] thermal pressure

[0960] Table 3C shows the turbidity, characterized by the high opalescence value (ranging from 7 NTU to 18 NTU) of the formulations in Table 3A during thermostress testing. No significant changes were observed in any sample after incubation at +40°C ± 2°C for 4 weeks.

[0961] Turbidity of formulations in Tables 3C–3A before and after 4 weeks of thermostressing at 40°C

[0962]

[0963]

[0964] Table 3D shows the percentage of deamidated forms in the thermostress tests of the formulations in Table 3A. At T0, no significant differences were observed in any samples compared to the standards, except for samples with high pH and relatively low ionic strength relative to API concentration—e.g., sample #15—which had a higher % deamidated form value than other samples. This may be attributed to the synergistic effect of high protein concentration, high pH, ​​and low ionic strength. Meanwhile, increasing pH at 4 weeks of thermostress at +40℃ ± 2℃ was associated with an increase in % deamidated forms. Overall, ionic strength and protein concentration had little effect on the percentage of deamidated forms. A pH range of 5.0–6.2 appears to be optimal, while formulations with pH between 7.1 and 7.5 performed poorly in this case.

[0965] Table 3D – % Deamidation Formation of the Formulations in Table 3A Before and After 4 Weeks of Heat Stress at 40°C

[0966]

[0967]

[0968] Figure 5 and Figure 6Together, they indicate that ionic strength and protein concentration have little effect on the % LMW material after heat stress, while pH has a significant effect. In this case, the pH range between 5.3 - 6.7 is clearly optimal. However, low ionic strength is associated with fragmentation at similar pH levels.

[0969] Figure 7 and Figure 8 Together, they indicate that the ionic strength should increase with the increase in protein concentration to avoid aggregate formation and ultimately protein precipitation. Additionally, in the case of the % HMW material after heat stress, the pH between 5.0 - 6.6 is clearly optimal.

[0970] At time = 0, among all the materials monitored by CGE (under reducing conditions), there were no significant differences in purity between the samples, except for sample #19, which had a slightly increased main cleavage.

[0971] After 4 weeks of heat stress at 40 °C, all samples with pH > 7 or pH < 5.5 showed lower overall purity, independent of ionic strength or protein concentration. At low pH conditions (< pH 5.5), low pH (pH 5.0 and to a lesser extent pH 5.3) led to an increase in peak 7 impurities, and an increase in the main cleavage (peak 6) was also observed.

[0972] At time = 0, cIEF showed no significant differences between the samples. <000234{2}>

[0973] Figure 9 And data elsewhere indicate that the effects of ionic strength and protein concentration on isotypes are not significant after heat stress, while the isotype distribution is affected by pH. Data from Figure 9 indicate that a pH of 5.3 - 5.9 is optimal in the case of isotypes.

[0974] Table 3E shows the isotype clusters after 4 weeks of heat stress at 40 °C.

[0975] Table 3E - Isotype Clusters after 4 Weeks of Heat Stress at 40 °C

[0976]

[0977]

[0978]

[0979] Figure 10 and Figure 11 Together, they indicate that ionic strength and protein concentration have little effect on the oxidation level, and only pH above 7.0 has a significant effect on the oxidation level. However, in the case of heat - promoted oxidation, a pH within the range of 5.3 - 6.6 (most preferably 5.7 - 6.2) seems to be the most favorable.

[0980] Light pressure

[0981] Table 3F shows the %HMW of the substances before and after photostress. Although there appears to be a low level of aggregate and significant effect of ionic strength between pH 5.2 and 6.6, photostress increases the %HMW of all samples.

[0982] Table 3F – Light Pressure (7 hours, 765 W / m) 2 %HMW of substances before and after

[0983]

[0984]

[0985] cIEF measurements of the samples exposed to photopressure showed no significant difference compared to the reference standards, except for samples 8, 15, 16, 18, and 19.

[0986] Table 3G shows the same type of clusters after photopressure.

[0987] Table 3G – Photopressure (7 hours, 765 W / m) 2 The same type of cluster after)

[0988]

[0989]

[0990]

[0991] Figure 12 , Figure 13 and Figure 14 Together, these findings indicate that under photostress conditions, ionic strength, protein concentration, and pH do not significantly affect the percentage of oxidation.

[0992] in conclusion

[0993] Figure 15These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contour lines (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration: 20 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions at this IgG:TGFβR2 concentration are a pH of 5.4–6.1, e.g., pH 5.7, and an ionic strength of 40–60 mM NaCl, e.g., 40 mM NaCl. Therefore, for pharmaceutical compositions containing 10–30 mg / mL IgG:TGFβR2 fusion protein, a pH of 5.2–6.3 and a minimum ionic strength, e.g., 10 mM NaCl, preferably in the range of 10–80 mM NaCl, are required.

[0994] Figure 16 These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contours (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration: 40 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions for this concentration of IgG:TGFβR2 are a pH of 5.7–6.1, e.g., pH 5.9, and an ionic strength of 50–70 mM NaCl, e.g., 60 mM NaCl. Therefore, for pharmaceutical compositions containing 30–50 mg / mL IgG:TGFβR2 fusion protein, a pH of 5.5–6.5 and a minimum ionic strength, e.g., 20 mM NaCl, preferably in the range of 30–90 nM NaCl, are required.

[0995] Figure 17 These are 2D and 3D contour plots, where the desirable parameters (reflecting the balance of factors in the overall response assessment) are represented as contours (in the 2D plot) and surfaces (in the 3D plot) within a formulation space (with a fixed protein concentration: 60 mg / mL) having variable pH and ionic strength (given in mM NaCl). This indicates that the optimal conditions at this IgG:TGFβR2 concentration are pH 5.7–6.1, e.g., pH 5.9, and 130–150 mM NaCl, e.g., an ionic strength of 150 mM NaCl. Optimal conditions at this concentration of IgG:TGFβR2 were also found at pH 5.7–6.1 (e.g., pH 5.9) and ionic strength of 80–90 mM NaCl (e.g., 80 mM NaCl). Therefore, for pharmaceutical compositions containing 50-70 mg / mL IgG:TGFβR2 fusion protein, a pH of 5.5-6.5 and a minimum ionic strength, such as 50 mM NaCl, are required, preferably in the range of 60-200 nM NaCl.

[0996] Example 3B – Further Screening Study – Step 1B (Buffer Screening)

[0997] General Overview

[0998] Following the results of Example 3A, experiments were conducted to confirm those results and explore alternative buffers. Therefore, three types of buffers were selected to cover a pH range of 5.7–6.2. Ionic strength was optimized based on protein concentration, according to the results of Example 3A.

[0999] List of test formulations

[1000] Table 3H shows the 12 formulations tested in this specific screening, plus the reference formulation.

[1001] Table 3H – Test Formulations

[1002]

[1003]

[1004] Results and Analysis

[1005] Under thermal stress conditions (4 weeks at 40°C) and light stress conditions (765 W / m) 2 (The following 7 hours) The formulations listed in Table 3H were studied. The samples subjected to heat pressure were tested using the following methods, although only the methods indicated in bold showed significant differences:

[1006]

[1007] Simultaneously, the light-sensitive samples were tested using the following methods:

[1008] ●HMW, via SE-UPLC

[1009] ● Overview of similar types, via ICE3

[1010] ●Oxidized form, via RP-UPLC

[1011] ●Purity / LMW, via CGE-SDS (Red / NoRed)

[1012] The qualitative results combining thermal and photo-stress conditions are shown in Table 3I below.

[1013] Table 3I shows the qualitative results (pass or fail) of the thermal and photostress tests for all formulations in Table 3H.

[1014] Table 3I – Qualitative results of thermal and photostress tests on formulations combined with those in Table 3H (NT = Not tested)

[1015]

[1016]

[1017] in conclusion

[1018] The results showed that histidine was the most stable of the three tested buffers, including at high protein concentrations, although other buffers, particularly succinate buffers, were also suitable under appropriate conditions. Furthermore, these tests highlight the feasibility of operation at higher protein concentrations (e.g., 40 and 60 mg / ml).

[1019] Example 3C – Further Screening Study – Step 2A (Excipient Screening)

[1020] General Overview

[1021] Based on the results of Example 3B, excipient screening was performed, maintaining the buffer at 10 mM histidine and pH 5.9, while varying the ionic strength between 60 mM and 100 mM and the protein (bintrafuspalfa) concentration between 40-60 mg / mL. All formulations in this example also contained 5 mM methionine to mitigate the oxidative effects, which were self-evident in the previous analysis.

[1022] The excipients to be screened are:

[1023] ● Sugar polyols, such as mannitol, trehalose, and sorbitol;

[1024] ● Amino acids, such as arginine, lysine, proline, and glutamic acid; and

[1025] ● Surfactants, such as Tween 20 and Kolliphor 188

[1026] The relevant formulations will be stress tested in the following manner before further analysis:

[1027] ● Thermal stress (40°C for up to 12 weeks)

[1028] ● Photopressure (at 765W / m) 2 (7 hours later)

[1029] ●F / T pressure (3X cycle -25℃ to RT)

[1030] ●Mechanical pressure (after 3 days at 300 rpm)

[1031] List of test formulations

[1032] Table 3J shows the 48 formulations tested in this specific screening. Table 3J – Test Formulations

[1033]

[1034]

[1035]

[1036]

[1037]

[1038]

[1039] Results and Analysis

[1040] Under thermal stress conditions (40℃ for 4 and 8 weeks) and light stress conditions (765W / m) 2 The formulations listed in Table 3J were studied under 7 hours of freezing and thawing pressure (3 F / T cycles, -25°C to room temperature) and mechanical pressure (3 days at 300 rpm).

[1041] The following methods were used to test the thermally stressed samples:

[1042]

[1043] The optically stressed sample was examined using the following methods:

[1044]

[1045] The F / T and mechanically stressed samples are inspected using the following methods:

[1046] ●HMW, via SE-UPLC

[1047] ●Purity / LMW, via CGE-SDS (Red / NoRed)

[1048] thermal pressure

[1049] Figure 18 and Figure 19 The results showed that protein concentration had a significant effect on %HMW under thermostress, but 50 mM arginine and 100 mM lysine were the best performing "various excipients" over 4 weeks, while 100 mM arginine and 100 mM lysine were the best performing over 8 weeks of thermostress.

[1050] Figure 20 and Figure 21The results showed that sorbitol (protein concentrations of 40 and 50 mg / mL) and mannitol (protein concentration of 60 mg / mL) exhibited the highest main clipping wave, 100 mM arginine had the highest %LMW, trehalose had the lowest, and protein concentration had no significant effect after 4 weeks of thermal stress.

[1051] Figure 22 The results showed that trehalose and 50 mM arginine exhibited the lowest % deamidation under thermo-stress, while L-glutamic acid, lysine, and proline showed the highest values.

[1052] Figure 23 and 24 Together, the results showed that polysorbate 20 exhibited superior performance compared to Kolliphor 188, and sorbitol actually showed the lowest % oxidized form after 4 weeks of thermal stress. Meanwhile, 50 mM L-arginine appeared to show the highest oxidized form under 8 weeks of thermal stress (protein concentration was constant, as it showed no effect).

[1053] Figure 25 This indicates that proline is closest to the target % cluster 1, or 52%, under thermal stress (protein concentration is constant, as it shows no effect).

[1054] Figure 26 This indicates that mannitol and lysine exhibit a lower percentage of cluster 2 than the target under thermal stress, which is 12%.

[1055] Light pressure

[1056] Figure 27 The results showed that 100 mM arginine and lysine exhibited the lowest %HMW, and that protein concentration had a significant effect on %HMW under light stress conditions.

[1057] Figure 28 and Figure 29 Together, the results showed that 100 mM arginine, sorbitol, and mannitol (the latter two only showed the highest % main clipping at a protein concentration of 60 mg / mL), while 50 mM arginine showed the highest % LMW under photostress conditions.

[1058] Figure 30 This indicates that 100 mM arginine provides the lowest % oxidation under photostress conditions.

[1059] freeze-thaw pressure

[1060] Figure 31 This indicates that mannitol exhibits the highest %HMW, and that protein concentration has no significant effect on %HMW under freeze-thaw pressure.

[1061] Mechanical pressure

[1062] Figure 32 and Figure 33 Together, these studies show that polysorbate 20 is superior to kolliphor 188 (especially in combination with sorbitol, arginine and lysine) in preventing aggregation under mechanical stress.

[1063] Overall Desirability

[1064] The overall desirability of different excipients is calculated based on a balance of factors, stress tests, and results.

[1065] Figure 34 This indicates that formulations with protein concentrations of 40 or 50 mg / mL are generally more desirable than those with 60 mg / mL.

[1066] For formulations with a protein concentration of 40 mg / mL, the ideal polyol is trehalose, and the ideal amino acids are 50 mM arginine and lysine.

[1067] For formulations with a protein concentration of 50 mg / mL, the ideal polyol is trehalose, and the ideal amino acids are 50 mM arginine and lysine.

[1068] For formulations with a protein concentration of 60 mg / mL, the ideal polyol is trehalose, and the ideal amino acids are 50 mM arginine and lysine.

[1069] Figure 35 This indicates that, compared to kolliphor 188, polysorbate 20 is slightly more desirable under almost all conditions.

[1070] Example 3D – Step 2B (Excipient Combination and Fine-tuning)

[1071] General Overview

[1072] Based on the overall desirability results of step 2A, it was decided to continue using protein concentrations of 40 and 50 mg / mL and to study the synergistic effect between sugars and amino acids to prepare a formulation combining trehalose and arginine / lysine.

[1073] List of test formulations

[1074] Table 3K shows the 12 formulations tested in this specific screening (+reference).

[1075] Table 3K – Test Formulations

[1076]

[1077]

[1078] Results and Analysis

[1079] Under thermal stress conditions (40℃ for 4, 8, and 12 weeks) and light stress conditions (765W / m) 2 The formulations listed in Table 3K were studied under mechanical pressure (300 rpm for 3 days) for 7 hours.

[1080] The following methods were used to test the thermally stressed samples:

[1081]

[1082] The optically stressed sample was examined using the following methods:

[1083]

[1084] The mechanical pressure sample was examined using the following method:

[1085] ●HMW, via SE-UPLC

[1086] ●Purity / LMW, via CGE-SDS (Red / NoRed)

[1087] Table 3L shows the nano-DSC results for the 12 formulations (+reference) tested in this specific screening.

[1088] Table 3. Nano-DSC results for L-formulation

[1089]

[1090]

[1091] Table 3L shows that formulations #89 and #91 have higher T values. m The values ​​indicate that these formulations have higher conformational stability. Comparing #89 and #90, it appears that increasing the Tween 20 concentration produces a stabilizing effect.

[1092] Figure 36 The results indicate that the increase in %HMW was comparable in combinations containing L-arginine and L-lysine; slightly higher values ​​were observed in formulations with a protein concentration of 50 mg / ml, which included excipients (#96, #97); formulations containing only one excipient (#100 and #101) achieved even higher values, while formulations containing EDTA showed a larger increase. The results for formulations #89–#95 were superior to those in 2A.5 of Table 2A (at the same protein concentration).

[1093] Figure 37 This indicates that the increase in %LMW is comparable in almost all formulations, with slightly higher values ​​observed in formulations containing EDTA.

[1094] Figure 38 This indicates that there was no significant difference in oxidation after 13 weeks of thermal stress, with the value being slightly lower for formulations containing only EDTA.

[1095] Figure 39 This indicates that all formulations exhibit similar deamidation effects after thermostressing.

[1096] Figure 40 This indicates that the purity of all formulations has decreased slightly, although the purity of each formulation is comparable.

[1097] Figure 41 This indicates that the main clipping peak (peak 6) increased in all formulations after thermostressing, with the highest observed increases observed in formulations containing EDTA (#98) and formulations containing only trehalose and a low amount of NaCl (#99 and reference). These identical formulations are also characterized by higher values ​​for impurities in peak 7.

[1098] Figure 42 The results showed a higher %HMW increase observed after applying photostress to the formulation: the formulation had lysine at a higher protein concentration of 50 mg / ml; contained EDTA; and had only one excipient.

[1099] Figure 43 The results showed that % oxidation increased in all formulations after exposure to photostress, with slightly higher values ​​observed in formulations containing EDTA and slightly lower values ​​observed in formulations containing only trehalose (#101).

[1100] Figure 44 This indicates that there was no significant difference in LMW levels among all formulations after photopressure.

[1101] Figure 45 This indicates that there was no significant change in purity after photopressure, except for formulation #99, which showed a slightly larger decrease after pressure.

[1102] Figure 46 This indicates that the level of the main clipping wave did not change significantly after optical pressure.

[1103] Figure 47 This indicates that %HMW changed only very slightly after mechanical pressure, although the initial value was relatively high for formulations containing EDTA, and a more significant increase was observed in formulations #91 and #95.

[1104] Figure 48 This indicates that %LMW did not change significantly after mechanical pressure, and only a moderate increase in LMW was observed in formulations #89 and #94.

[1105] Figure 49 This indicates that formulation #95 showed a significant decrease in purity after mechanical pressure, while other formulations showed only slight changes.

[1106] Figure 50 This indicates that the percentage of the main clipping wave did not change significantly after mechanical pressure, except for formulation #95, which contains the highest amount of lysine.

[1107] in conclusion

[1108] Based on the data collected in this formulation screening, the best-performing formulations under all conditions were those with excipient combinations, particularly those with trehalose combined with L-arginine. Two formulations with a protein concentration of 50 mg / ml showed performance comparable to those at 40 mg / ml, indicating that higher concentrations are indeed feasible. Table 2A.5, containing 40 mg / mL bintrafusp alfa and 60 mM NaCl, used for comparison in the thermostress test, appears to be largely comparable to formulations with excipient combinations.

[1109] Overall, formulations #2A.5, #89, #91 and #96 in Table 2A are considered particularly promising candidates.

[1110] Example 4 – Further Formulation Studies

[1111] General Overview

[1112] The overall plan for this embodiment is to initially screen samples of bintrafusp alfa at a concentration of 20-25 mg / mL to evaluate pH, buffers, and excipients. Subsequently, prior to subsequent surfactant screening, high-concentration and short-term stability tests were performed on the selected formulations, with short-term stability tests ultimately conducted on 4-5 high-concentration formulations.

[1113] List of test formulations

[1114] Table 4A shows 20 formulations (F1-F20), including the reference formulation (F1) to be tested in this specific screening.

[1115] Table 4A – Test Formulations

[1116]

[1117]

[1118]

[1119] Results and Analysis

[1120] Store and test formulations according to the following summary listed in Table 4B (tests performed are marked with "x").

[1121] Table 4B

[1122]

[1123]

[1124] In this case, T0 is at time 0; T-FT is after 3 freeze-thaw cycles ( Figure 51 T-2w and T-4w indicate the temperature after 2 weeks at 25℃ or 40℃, respectively.

[1125] Figure 52 It is a bar chart showing how the pH of all formulations F1-F20 changed at different time points during the pressure test, including (the bars for each formulation listed from left to right): time = 0; after 3 FT cycles; after 2 weeks at 25°C, after 2 weeks at 40°C, after 4 weeks at 25°C, and after 4 weeks at 40°C.

[1126] The target pH values ​​for each formulation are listed in Table 4A. Figure 52 This illustrates the change.

[1127] For all formulations, the target pH (±0.1) is reached at T0.

[1128] After 4 weeks of storage, the pH value of most formulations did not change.

[1129] For the three formulations with a protein concentration of 50 mg / ml, lower pH values ​​were detected:

[1130] ●F12 (His buffer pH 5.7) can be stored at 40°C for 4 weeks.

[1131] ●F17 (unbuffered, pH 5.7) can be stored at 40°C for 4 weeks.

[1132] ●F19 (containing sorbitol, pH 6.0) can be stored at 25°C for 2 and 4 weeks.

[1133] Figure 53 It is a bar chart showing the weight permeable molar concentration of each of the formulations F1-F20.

[1134] Except for F15 (which was found to have a lower weight osmolar concentration of 254 mOsmol / kg), all formulations achieved weight osmolar concentrations within the specification range of 260-340 mOsmol / kg (green line).

[1135] Figure 54 It is a bar chart showing how the turbidity of all formulations F1-F20 changed at different time points during the pressure test, including (the bars for each formulation listed from left to right): time = 0; after 3 FT cycles; after 2 weeks at 25°C, after 2 weeks at 40°C, after 4 weeks at 25°C, and after 4 weeks at 40°C.

[1136] Turbidity appears to be dependent on protein concentration—higher turbidity values ​​were detected in samples with higher protein concentrations. Turbidity values ​​for most formulations typically remained unchanged, but increases of 0.5 to 2.0 were observed in the following formulations:

[1137] ●F3 (His buffer pH 5.7) at T4w-25℃

[1138] ●F13 (His buffer pH 6.0) at T2w-25℃ and T2w-40℃

[1139] ●F17 (unbuffered, pH 5.7) at T2w-25℃ and T4w-25℃

[1140] For the following formulations, an increase of 4.1 to 7.4 was observed:

[1141] ●F19 (containing sorbitol, pH 6.0) at T2w-25℃ and T4w-25℃

[1142] Visual inspections are performed and reported according to the codes listed in Tables 4C and 4D.

[1143] Table 4C – Particle Visibility Code

[1144] Visible particle fraction describe 0 No particles visible within 5 seconds 1 The particles are almost invisible within 5 seconds. 2 A moderate number of particles are visible within 5 seconds. 10 A large number of particles are directly visible

[1145] Table 4D – Codes of Visible Features

[1146]

[1147] The visual inspection results under the various pressure conditions and time points mentioned above are listed in Tables 4E, 4F and 4G below.

[1148] Table 4E – Inspection results after time = 0 and 3 FT cycles

[1149]

[1150]

[1151] Table 4F – Visual inspection results after two weeks at 25°C and 40°C

[1152]

[1153]

[1154] Table 4G – Visual inspection results after four weeks at 25°C and 40°C

[1155]

[1156]

[1157] The results of the analysis based on Table 4A are shown in Table 4H.

[1158] Tables 4H and 4A show the analytical results of the formulations before and after pressure (the classification of each result is shown in superscript according to Table 4I).

[1159]

[1160]

[1161]

[1162]

[1163]

[1164]

[1165]

[1166]

[1167]

[1168]

[1169]

[1170]

[1171] The analysis results in Table 4H are classified based on the thresholds defined in Table 4I (see the superscript numbers in Table 4H).

[1172] Table 4I – Thresholds applicable to classifications in Table 4H

[1173]

[1174]

[1175] Regarding the UV spectral (SoloVPE) analysis used to determine the concentration of bintrafusp alfa, it was noted that all target concentrations were reached at time = 0. No significant changes were observed after pressure was applied.

[1176] For all formulations, the detected viscosity was relatively low, approximately 1.0–1.7 mPa*s, with higher viscosity values ​​(1.6–1.7 mPa*s) observed for formulations containing a protein concentration of 50 mg / ml (F9–F20).

[1177] At T0, the smallest subvisible particles were detected in all formulations. After 2 and 4 weeks, an increase in subvisible particles was observed in F17 (unbuffered system, pH 5.7) and F19 (sorbitol-containing formulations) stored at 25°C, and in F8 (sorbitol-containing formulations) stored at 40°C. After 4 weeks of storage at 25°C and 40°C, F5 showed the largest increase in visible particles. For most formulations, low particle concentrations (less than 1,000 particles ≥5 μm) were detected.

[1178] Regarding the HP-SEC results, it was noted that the LMW shoulder of F19 was evolving after 4 weeks of storage at 25°C. However, unlike HMW, LMW was not integrated into the overall monomer content results. Generally, for all formulations, a decrease in monomer content and an increase in HMW content were observed after storage at 25°C and 40°C for up to 4 weeks. Formulations containing arginine showed less decrease in monomer content compared to other formulations with higher protein concentrations. At higher protein concentrations, pH 6.0 appeared to be superior to pH 5.5 and 5.7 (testing His as a buffer system). The comparison between F16 and F20 suggests that more arginine and less NaCl may have a stabilizing effect on the protein.

[1179] Regarding deamidation (assessed by IEX-HPLC, peak 2 content % – peak 2 is the largest peak, any decrease in peak 2 size indicates deamidation), for all formulations, the relative peak area for peak 2 at T0 was measured to be >93%. A decrease in the relative peak area of ​​peak 2 was observed in all formulations after storage, which was more pronounced at 40°C than at 25°C – thus, an increase in peak area was observed in the “LC Deam peak” group (which is a combination of all peaks distinct from peak 2).

[1180] Following the FT, a slight increase in the relative peak area of ​​the "LC Deam peak" was observed, which is comparable to storage at 25°C for 2 weeks. For higher concentration formulations, the most promising results were obtained in the following aspects:

[1181] ●25mg / ml protein concentration:

[1182] His buffer systems at pH 5.5 and 5.7 (F2 and F3)

[1183] o Preparations containing arginine and sorbitol (F7 and F8)

[1184] ● 50 mg / ml protein concentration: His buffer system pH 5.5 and 5.7 (F11 and F12)

[1185] Figure 55 This is the T-wave pattern of nano-DSC traces of all formulations F1-F20 at different time points during stress testing. mThe bar chart shows how the temperature of the two peaks changes, including (the bars for each formulation listed from left to right): Time = 0; after 3 FT cycles; 2 weeks at 25°C, 2 weeks at 40°C, 4 weeks at 25°C, and 4 weeks at 40°C.

[1186] Figure 56 This shows the onset temperature T of the nano-DSC traces of all formulations F1-F20 at different time points during the pressure test. m The bar chart showing how the changes are (bars listed from left to right for each formulation): Time = 0; after 3 FT cycles; after 2 weeks at 25°C, after 2 weeks at 40°C, after 4 weeks at 25°C, and after 4 weeks at 40°C.

[1187] Figure 55 and Figure 56 Together, they indicate the T values ​​of all formulations. m Both values ​​were above 66°C, and no significant changes were observed during pressure or storage.

[1188] In terms of the overall characteristics of the same type, there were no significant changes. At time = 0, the following relative peak areas were detected:

[1189] →Cluster 1 is approximately 56%

[1190] →Cluster 2 approximately 11%

[1191] →Cluster 3 is approximately 21%

[1192] →Cluster 4 is approximately 12%

[1193] Freeze-thaw cycles appear to have no effect on the isotype profile of the sample. After storage at 40°C for 4 weeks, please note the following:

[1194] →The relative peak area of ​​cluster 2 decreases.

[1195] →The relative peak area of ​​cluster 4 increases

[1196] cGE was performed under both reducing and non-reducing conditions, and the results are listed in Table 4H. Under reducing conditions, all formulations yielded very similar results at time = 0 (the difference between the maximum and minimum purity [%) was 0.6%). The purity of all formulations decreased after 4 weeks of storage at 40°C, although this decrease was least significant for F16, F18, and F20, and most significant for F1, F8, F12, and F17. The decrease in purity was mainly accompanied by an increase in the main clipping [%). In addition, although the content of other impurities increased, these had little effect on the overall purity of the formulations.

[1197] For cGE under non-reducing conditions, very similar results were observed for all formulations at time = 0 (the difference between the maximum and minimum corrected peak areas [%) of intact molecules was 1.1%). The relative peak areas of intact molecules decreased for all formulations after storage at 40°C for 4 weeks, although this decrease was least significant for F5, F6, F14, and F15, and most significant for F2, F19, and F12. The decrease in the corrected peak area of ​​intact molecules was accompanied by an increase in the relative peak area of ​​LMW substances (T4w_40°C).

[1198] Oxidation measurements by RP-UPLC are recorded in Table 4H. After storage at 40°C for 4 weeks, most samples showed a decrease in intact Met516 content, while the content of oxidized Met516 increased, but this was most pronounced in F4, F9, and F15.

[1199] in conclusion

[1200] At time = 0, all formulations F1-F20 reached their target pH, concentration, and weight osmolality. A small amount of visible and subvisible particles were observed, and all formulations exhibited relatively low viscosity. HP-SEC showed a 98% concentration of the dominant peak in all formulations. cGE showed approximately 94% purity for all formulations under reducing conditions and greater than 96% purity under non-reducing conditions.

[1201] Freeze-thaw (FT) pressure had no significant effect on the stability of the formulation. Furthermore, after FT pressure, pH, concentration, number of visible and subvisible particles, turbidity, and monomer content remained essentially unchanged.

[1202] Samples stored at 25°C and 40°C for up to 4 weeks exhibited the following characteristics:

[1203] ● pH remains essentially unchanged

[1204] ● Turbidity values ​​increased; samples stored at 25°C showed higher values ​​compared to samples stored at 40°C.

[1205] ●More visible particles were observed after storage at 25°C and 40°C for 4 weeks.

[1206] ● Protein concentration remains unchanged

[1207] ● Relatively large amounts of subvisible particles were observed in F5, F8, F17, and F19.

[1208] ● Formulations containing arginine show less decrease in monomer content over time (HP-SEC)

[1209] ●cGE: The purity (%, under reducing conditions) and the content of intact molecules (%, under non-reducing conditions) of all formulations decreased after storage at 40°C for 4 weeks → F12 performed the worst in these two parameters.

[1210] Finally, the percentage oxidation measured by RP-UPLC showed that, at time = 0, the intact Met516 was between 96 and 97% for all formulations, and a decrease in intact Met516 and an increase in oxidized Met516 were observed after storage at 40°C for 4 weeks.

[1211] Formulations containing 25 mg / mL bintrafusp alfa (F2-F8) appeared to perform best at pH 5.5, although it was noted that citrate buffer performed best at pH 6.0. Beneficial effects were also observed at this concentration when arginine was included in the formulation.

[1212] Formulations containing 50 mg / mL bintrafusp alfa (F9-F20) appear to perform best at pH 5.5, although at this concentration, histidine buffers perform best relative to other buffers at pH 6.0. Arginine is also considered beneficial at lower concentrations.

[1213] Further testing is currently underway for the following promising candidates, F13, F16, F20 and F21-F23, as detailed in Table 4J.

[1214] Table 4J – Candidate formulations generated from this example

[1215]

[1216]

[1217] Example 5 – Further Formulation Studies

[1218] General Overview

[1219] To further examine the suitability of ion tension modifiers in combination with sugar and / or amino acid components to stabilize IgG:TGFβR fusion proteins, different IgG:TGFβR fusion proteins were tested in such formulations. Specifically, IgG:TGFβR fusion proteins with different IgG classes, different complementary sites, and different TGFβR moieties were tested.

[1220] List of IgG:TGFβR fusion proteins tested

[1221] The following three anti-PD-L1(IgG):TGFβR2 fusion proteins were tested:

[1222] ●bintrafusp alfa (the light chain and heavy chain sequences correspond to SEQ ID NO:7 and SEQ ID NO:8, respectively)

[1223] ● Fusion protein 2 (the light chain and heavy chain sequences correspond to SEQ ID NO:33 and SEQ ID NO:34, respectively)

[1224] ● Fusion protein 3 (the light chain and heavy chain sequences correspond to SEQ ID NO:15 and SEQ ID NO:18, respectively)

[1225] List of test formulations

[1226] The formulations shown in Table 5A were compared (all formulations additionally contain 40 mg / ml fusion protein, 10 mM histidine buffer, 0.05% Tween 20, 5 mM methionine, pH 5.9):

[1227] Table 5A Test Formulations

[1228]

[1229]

[1230] Results and Analysis

[1231] The formulations listed in Table 5A are the subjects of pressure stability studies (at 40°C for 4 weeks).

[1232] Figure 57 is a bar chart showing the percentage of high molecular weight (%HMW) substances by SE-UPLC. Consistent with the above results and further confirmed by statistical evaluation, arginine can reduce aggregation, and increasing the amount of NaCl can also stabilize proteins (even in the absence of arginine).

[1233] Figure 58 is a bar chart showing the percentage of low molecular weight (%LMW) substances as measured by CGE-NRED. Consistent with earlier results and further confirmed by statistical evaluation, while trehalose prevents fragmentation, arginine tends to increase fragmentation, although the effect of arginine can be masked by the stabilizing effect of trehalose.

[1234] Table 5B shows the unfolding temperatures measured by micro-DSC. No significant changes in unfolding temperature were observed for any of the formulations tested.

[1235] Table 5 shows the micro-DSC results for formulation B.

[1236]

[1237]

[1238] No materials remaining for measurement.

[1239] The tertiary structure of the samples was measured by fluorescence, and no loss of tertiary structure was observed in any sample.

[1240] in conclusion

[1241] The collected data on each fusion protein were further analyzed using a consensual graph.

[1242] For example, Figure 59 3D contour plots are shown, where the desirability parameters (reflecting the balance of factors in the overall response assessment) are represented as surfaces in formulation space, where trehalose concentration and ionic strength (given in mM NaCl) are variable for the tested anti-PD-L1(IgG):TGFβR2 fusion protein. In these plots, arginine is maintained at a constant concentration of 100 mM because this was found to be the most desirable (see also...). Figure 60 and 61 ).

[1243] Figure 60 3D contour plots are shown, where the desirability parameters (reflecting the balance of factors in the overall response assessment) are represented as surfaces within the formulation, where arginine concentration and ionic strength (given in mM NaCl) are variable for the tested anti-PD-L1(IgG):TGFβR2 fusion protein. In these plots, trehalose was maintained at a constant concentration of 200 mM because this was found to be the most desirable (see also...). Figure 59 and 61 ).

[1244] Figure 61 3D contour plots are shown, where the desirability parameters (reflecting the balance of factors in the overall response assessment) are represented as surfaces within the formulation, where arginine and trehalose concentrations are variable for each anti-PD-L1(IgG):TGFβR2 fusion protein tested. In these plots, the ionic strength (given as mM NaCl) is maintained at a constant concentration of 100 mM, as this was found to be the most desirable (see also...). Figure 59 and 60 ).

[1245] Overall, it was found that using increased amounts of ionic strength, amino acid composition, and disaccharide for the tested anti-PD-L1(IgG):TGFβR2 fusion protein was desirable, as reflected in the desirability plot, indicating that using increased concentrations of all three components is preferable.

[1246] Summary of Conclusions

[1247] Example 1 shows that:

[1248] ● The pH of the pI that is relatively close to one of the key protein domains (i.e., the TGFβR2 part) is surprisingly favorable for protein solubility and stability.

[1249] ●Minimum ionic strength (suitably provided by sodium chloride) is preferred to promote protein dissolution.

[1250] ●At least from the perspective of protein solubility, higher ionic strength is usually required to support high protein concentrations.

[1251] ● Freeze-drying helps with the long-term storage of formulations, and freeze-dried formulations can be easily reconstituted.

[1252] ● Trehalose helps with freeze drying, but alternatives may be considered.

[1253] ● If excessive amounts of sodium chloride are used to achieve higher ionic strength, such as to support higher protein concentrations, lyophilization becomes more challenging. In such cases, alternative ionic strength providers may be considered.

[1254] Example 2 shows that:

[1255] ●The formulation of Example 1 is practically feasible from the perspective of long-term storage stability when prepared in liquid form.

[1256] ● It can increase protein concentration, especially in liquid formulations, while maintaining its activity in terms of long-term storage stability.

[1257] ● Formulations with higher concentrations of the relevant proteins often benefit from higher ionic strength (e.g., higher NaCl concentrations).

[1258] Surprisingly, NaCl also supports protein solubility and protein stability.

[1259] ●Histidine is clearly a good buffer for these formulations, although alternatives may be considered.

[1260] Example 3 shows that:

[1261] ● Higher protein concentrations typically require higher ionic strength, which reinforces previous findings.

[1262] ● Higher protein concentrations may benefit from slightly higher pH, although still closer to the pI of the TGFβR2 group than is typically expected.

[1263] ●A variety of buffers are acceptable, provided the pH is suitable for both the protein and the buffer, although the optimal pH may vary slightly between different buffers. Histidine buffers appear to offer the best performance.

[1264] ● For different pressure conditions (heat, light, mechanical, freeze-thaw), some excipients are superior to others, which may vary due to different factors such as fragmentation, conformational stability, aggregation, homologation, deamidation, oxidation, and turbidity.

[1265] ● Arginine, trehalose, and lysine appear to be the best performing excipients, although their relative ranking varies with protein concentration.

[1266] ● Trehalose generally outperforms other sugar components, such as mannitol and sorbitol.

[1267] ●Arginine has been shown to be an excellent stabilizer, especially in preventing aggregation.

[1268] ● Polysorbate 20 appears to be slightly superior to the Kolliphor 188 surfactant, especially in reducing aggregation.

[1269] ●Overall, combinations of trehalose and arginine in different relative molar ratios have proven advantageous.

[1270] Example 4 shows that:

[1271] ● Arginine may (partially) replace NaCl as a source of ionic strength.

[1272] ● Unbuffered systems seem to be less advantageous.

[1273] ● For high protein concentrations, a slightly higher pH seems to work better.

[1274] Example 5 further confirms that increased amounts of ionic strength, amino acid composition, and sugars are beneficial for stabilizing the IgG:TGFβR fusion protein. sequence list <110> Ares Trading Co., Ltd. GlaxoSmithKline Intellectual Property (No. 4) Ltd. <120> IgG:TGFβRII fusion protein composition <130> P19-242 WO-PCT <150> EP19219008.0 <151> 2019-12-20 <150> EP20186428.7 <151> 2020-07-17 <150> EP20194928.6 <151> 2020-09-07 <160> 35 <170> BiSSAP 1.3.6 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 1 Ser Tyr Ile Met Met 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 2 Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 3 Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 4 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 5 Asp Val Ser Asn Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 6 Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val 1 5 10 <210> 7 <211> 216 <212> PRT <213> Artificial sequence <220> <223> From the Fab Library <400> 7 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 8 <211> 607 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val 465 470 475 480 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 485 490 495 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 500 505 510 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 515 520 525 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 530 535 540 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 545 550 555 560 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 565 570 575 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 580 585 590 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 9 <211> 592 <212> PRT <213> Homo sapiens <400> 9 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp 20 25 30 Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn 35 40 45 Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr 50 55 60 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 65 70 75 80 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 85 90 95 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 100 105 110 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 115 120 125 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 130 135 140 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 145 150 155 160 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 165 170 175 Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val 180 185 190 Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile 195 200 205 Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser 210 215 220 Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His 225 230 235 240 Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys 245 250 255 Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp 260 265 270 Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu 275 280 285 Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe 290 295 300 Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser 305 310 315 320 Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu 325 330 335 Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe 340 345 350 His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser 355 360 365 Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala 370 375 380 His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile 385 390 395 400 Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu 405 410 415 Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr 420 425 430 Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg 435 440 445 Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val 450 455 460 Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp 465 470 475 480 Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu 485 490 495 Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met 500 505 510 Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe 515 520 525 Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu 530 535 540 Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala 545 550 555 560 Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser 565 570 575​​​​​​​​​​​​​​​Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565 <210> 11 <211> 136 <212> PRT <213> Homo sapiens <400> 11 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr<​​​​​​​​​​Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 12 <211> 117 <212> PRT <213> Homo sapiens <400> 12 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 1 5 10 15 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 20​​​​​Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 50 55 60 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 65 70 75 80 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 85 90 95 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 100 105 110 Thr Ser Asn Pro Asp 115 <210> 13 <211> 115 <212> PRT <213> Homo sapiens <400> 13 Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr 1 5 10 15 Cys Asp Asn Gln Lys ...

Claims

1. A pharmaceutical composition comprising 5-45 mg / mL of an IgG:TGFpR fusion protein, 30-60 mM of an ionic tonicity modifier, 65-170 mM of a disaccharide, 0.25-0.75 mg / mL of a surfactant, a buffer system, optionally 1-10 mM of an antioxidant, and optionally 10-95 mM of an amino acid component; wherein the ionic tonicity modifier is sodium chloride; the disaccharide is trehalose; the surfactant is polysorbate 20; the buffer system does not comprise an acetate buffer, and the buffer system is a histidine buffer system; the amino acid component is arginine; the antioxidant is methionine; and the composition has a pH of 5.0 to 6.3; and the light chain sequence and the heavy chain sequence of the IgG:TGFpR fusion protein correspond to (1) SEQ ID NO: 7 and SEQ ID NO: 8, (2) SEQ ID NO: 15 and SEQ ID NO: 17, (3) SEQ ID NO: 15 and SEQ ID NO: 18, or (4) SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

2. The pharmaceutical composition of claim 1, wherein the composition is selected from the group of the following compositions: a. A pharmaceutical composition, characterized in that a pH of 5.3-5.7 and comprising 5-15 mg / mL IgG:TGFpR fusion protein, 5-15 mM buffer system, 30-50 mM ionic tonicity modifier, 150-170 mM disaccharide, 0.3-0.7 mg / mL surfactant, and optionally 2-8 mM antioxidant; b. A pharmaceutical composition, characterized in that a pH of 5.3-5.7 and comprising 35-45 mg / mL IgG:TGFpR fusion protein, 5-15 mM buffer system, 50-60 mM ionic tonicity modifier, 150-170 mM disaccharide, 0.3-0.7 mg / mL surfactant, and optionally 2-8 mM antioxidant; c. a pharmaceutical composition characterized by a pH of 5.7-6.1 and comprising 35-45 mg / mL IgG:TGFpR fusion protein, 5-15 mM buffer system, 50-60 mM ionic tonicity modifier, 90-110 mM disaccharide, 40-60 mM amino acid component, 0.3-0.7 mg / mL surfactant, and optionally 2-8 mM antioxidant; d. A pharmaceutical composition, characterized by a pH of 5.7-6.1 and comprising 35-45 mg / mL IgG:TGFpR fusion protein, 5-15 mM buffer system, 50-60 mM ionic tonicity modifier, 65-85 mM disaccharide, 65-85 mM amino acid component, 0.3-0.7 mg / mL surfactant, and optionally 2-8 mM antioxidant; e. A pharmaceutical composition characterized by a pH of 5.5 and comprising 10 mg / mL IgG:TGFpR fusion protein, 10 mM histidine, 40 mM sodium chloride, 159 mM trehalose, 0.5 mg / ml polysorbate 20, and 5 mM methionine; f. A pharmaceutical composition characterized by a pH of 5.5 and comprising 40 mg / mL IgG:TGFpR fusion protein, 10 mM histidine, 60 mM sodium chloride, 159 mM trehalose, 0.5 mg / ml polysorbate 20, and 5 mM methionine; and g. A pharmaceutical composition characterized by a pH of 5.9 and comprising 40 mg / mL IgG:TGFpR fusion protein, 10 mM histidine, 60 mM sodium chloride, 100 mM trehalose, 50 mM arginine, 0.5 mg / ml polysorbate 20, and 5 mM methionine.

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