IgG:TGFβRII fusion protein composition

Abstract

The present invention relates to pharmaceutical compositions, particularly pharmaceutical compositions containing an IgG:TGFβRII (e.g., anti-PD-L1:TGFβ-inhibitory) fusion protein. The invention also relates to, inter alia, methods of making the compositions, kits comprising the compositions, packages comprising the compositions, methods of making the packages, and methods of treatment, particularly cancer treatment, using the compositions and / or packages.

Description

[Technical Field]

[0001] Introduction The present invention relates to pharmaceutical compositions, particularly pharmaceutical compositions containing an IgG:TGFβRII fusion protein. The invention also relates to, inter alia, methods of making the compositions, kits containing the compositions, containers or drug delivery devices containing the compositions, methods of making the containers or drug delivery devices, and methods of treatment, particularly cancer treatment, using the compositions and / or containers or drug delivery devices. [Background technology]

[0002] background 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 transforming growth factor beta receptor type II (TGFβRII) as a "trap" that neutralizes TGFβ into a single molecule. Specifically, this protein is a heterotetramer consisting of two immunoglobulin light chains of an anti-PD-L1 antibody and two heavy chains containing the anti-PD-L1 antibody heavy chain genetically fused to the extracellular domain of human TGFβRII via a flexible glycine-serine linker (see Figure 1). This anti-PD-L1 / TGFβ trap molecule is designed to target two major mechanisms of immune suppression in the tumor microenvironment and therefore may be used in the treatment of cancer or inhibition of tumor growth.

[0003] It is an object of the present invention to provide a viable pharmaceutical composition of an IgG:TGFβRII fusion protein. The unpredictability inherent in the art of formulating biologics, particularly antibody- or antibody fragment-containing biologics, makes the discovery of such a viable pharmaceutical composition difficult, since most formulations of a given biopharmaceutical (if such formulation is chosen at all) are unstable over long periods of time and / or under stress conditions due to the various degradation pathways to which biologics, particularly aqueous formulations, are susceptible. Degradation factors may include, for example, one or more of the following (typically two or more, and potentially three or more): Physical effects, e.g.: ○ Inappropriate inhibition of aggregation of related protein molecules; o Inappropriate inhibition of precipitation; Inadequate inhibition of the adsorption of relevant protein molecules at the water-air interface or at the interface with any packaging material; o Improper regulation of osmolality; Chemical action, e.g.: o Improper regulation of oxidation; Inappropriate inhibition of photo-oxidation; Inadequate inhibition of the hydrolysis of ester bonds, leading to the formation of acid, aldehyde and peroxide products, thus affecting the stability of the fusion protein; o Improper stabilization and maintenance of pH; ○ Inappropriate inhibition of protein fragmentation; o Inappropriate inhibition of protein unfolding.

[0004] Any, some, or all of the aforementioned factors can lead to either an unviable drug product (which may be unsafe for use in medical treatment) or a drug product whose viability is variable and unpredictable, especially considering the various stresses (agitation, freeze-thaw, heat, light) to which different batches of drug product may be exposed during manufacturing, transportation, and storage.

[0005] The present invention preferably seeks to address one or more of the stability problems discussed above, and in doing so, to provide a viable pharmaceutical formulation. Summary of the Invention

[0006] Summary of the Invention In some embodiments, a pharmaceutical composition containing an IgG:TGFβR fusion protein is provided. The pharmaceutical composition of the present invention may contain, consist of, or exclude any, some, or all of the components described herein (e.g., including buffers, surfactants, sugar components, amino acid components, tonicity agents, antioxidants, chelating agents; or, in fact, excluding any of the foregoing), preferably in any relevant amounts as described herein, and / or may 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.

[0007] In some embodiments, a container or drug delivery device is provided that contains or comprises a pharmaceutical composition as defined herein. Such a drug delivery device can be, for example, a vial, an ampoule, a syringe, a pre-filled syringe, an injection pen (e.g., essentially a combined syringe), an autoinjector, or an intravenous infusion bag, or a package / container that includes any of the foregoing.

[0008] In some embodiments, a kit of parts is provided that includes a drug delivery device, a pharmaceutical composition as defined herein, and optionally a set of instructions for administering the pharmaceutical composition (e.g., intravenously, subcutaneously).

[0009] In some embodiments, a method for producing a pharmaceutical composition is provided, the method comprising admixing an IgG:TGFβR fusion protein together with one or more pharmaceutically acceptable excipients and / or carriers.

[0010] In some embodiments, there is provided a method of treating a disease or medical disorder in a patient in need of such treatment, the method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0011] In some embodiments, there is provided a pharmaceutical composition as defined herein for use in treating a disease or medical disorder in a patient in need of such treatment.

[0012] In some embodiments, there is provided a use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a disease or disorder.

[0013] In some embodiments, there are provided methods of treating a disease or medical disorder, pharmaceutical compositions for use in treating a disease or medical disorder, and uses of pharmaceutical compositions in the manufacture of a medicament for the treatment of a disease or disorder, as defined herein, wherein the disease or medical disorder is a PD-L1-associated disease, a TGFβ-associated disease and / or a proliferative disease or disorder, preferably cancer.

[0014] In some embodiments, the treating or therapy involves combination therapy, whereby the pharmaceutical composition is administered in combination with one or more other pharmaceutically or biopharmaceutical active substances(s); wherein the combination therapy involves simultaneous, sequential, or separate administration of the individual components of the therapy. In some embodiments, the additional pharmaceutically or biopharmaceutical active substances may be present within any of the pharmaceutical compositions as defined herein.

[0015] All of the foregoing methods of treatment, composition(s) for use, and use of the composition(s) in the manufacture of a medicament are equally applicable to associated containers, drug delivery devices, and kits incorporating the composition(s).

[0016] Any feature, including any suitable and preferred feature, described in connection with a particular aspect of the invention may also be a feature, including any suitable and preferred feature, of any other aspect of the invention, unless incompatible therewith. [Brief explanation of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the present invention, and to show how embodiments thereof may be put into effect, reference is now made, by way of example, to the following schematic drawings:

[0018] [Figure 1A] Figure 1A shows half of the sequence of the bintrafsalpha fusion protein, which includes the light chain (designated VL and CL regions) and heavy chain (designated VH, CH1, CH2, and CH3 regions) connected to the extracellular domain (ECD) of TGFβ receptor II via the designated linkers.

[0019] [Figure 1B]Figure IB shows half the sequence of a bintrofusalpha fusion protein, comprising a light chain (designated VL and CL regions) and a heavy chain (designated VH, CH1, CH2, and CH3 regions) connected to the extracellular domain (ECD) of TGF-beta receptor II via the designated linker. The overall sequences of each of the light chain, heavy chain, linker, and extracellular domain of TGF-beta receptor II are identical for both Figures 1A and 1B, and Figures 1A and 1B are intended to correspond, with respect to the overall sequence, to "bintrofusalpha" as defined in the definitions section of this application, designated CAS Registry Number 1918149-01-5. Figure 1B is the same as Figure 1A, except that the first three amino acids (PCP) of the CH2 region in Figure 1A are the last three amino acids (PCP) of the hinge region in Figure 1B; and the last three amino acids (GQP) of the VL region in Figure 1A are the first three amino acids (GQP) of the CL region in Figure 1B. The assignment of specific sequences to specific regions, as shown in Figures 1A and 1B, is to facilitate these comparisons. For purposes of region-specific comparisons, the assignment of specific sequences to specific regions shown in Figure 1A or 1B may be used. In some embodiments, the assignment of specific sequences to specific regions shown in Figure 1B may be used.

[0020] [Figure 2] Figure 2 shows a graphical representation of the relationship between Tg' (°C) and NaCl concentration for formulations containing 8% trehalose (diamonds), 4% trehalose (squares), and 2% trehalose (triangles). From a practical standpoint during freeze-drying, Tg' values ​​below -40°C are undesirable.

[0021] [Figure 3] Figure 3 is a contour plot of the Tm3 unfolding temperature, expressed as a contour line (ranging from 69.98 to 72.72) within the formulation space (with ionic strength fixed at 95 mM NaCl), at various protein concentrations (Vintrofus alpha mg / mL) and pH.

[0022] [Figure 4] Figure 4 is a contour plot of the Tm3 unfolding temperature, expressed as a contour line (ranging from 69.98 to 72.72) within the formulation space (with pH fixed at pH 6.25), at various protein concentrations (Vintrofus alpha mg / mL) and ionic strengths (mM NaCl).

[0023] [Figure 5] Figure 5 shows a contour plot of %LMW species after 4 weeks at 40°C, expressed as contours (ranging from 5.71 to 12.35) within the formulation space (with ionic strength fixed at 95 mM NaCl) at various protein concentrations (Vintrofus Alpha mg / mL) and pH. This data was obtained by CGE non-reducing, i.e., CGE under non-reducing conditions.

[0024] [Figure 6] Figure 6 is a contour plot of %LMW species after 4 weeks at 40°C, expressed as contour lines (see contour lines at 6, 6.5, 7, and 7.5) within the formulation space (with pH fixed at 6.25) at various protein concentrations (Vintrofus Alpha mg / mL) and ionic strengths (mM NaCl). Data was obtained by CGE non-reducing.

[0025] [Figure 7] Figure 7 is a contour plot of % HMW species after 4 weeks at 40°C, expressed as a contour (ranging from 1.2 to 6.3) within the formulation space (with ionic strength fixed at 95 mM NaCl) at various protein concentrations (Vintrofus alpha mg / mL) and pH. This data was obtained by SE-UPLC.

[0026] [Figure 8]Figure 8 is a contour plot of % HMW species after 4 weeks at 40°C, expressed as contour lines (see contour lines at 1, 2, 3, and 4) within the formulation space (with pH fixed at 6.25) at various protein concentrations (Vintrofus alpha mg / mL) and ionic strengths (mM NaCl). This data was obtained by SE-UPLC.

[0027] [Figure 9] Figure 9 shows a contour plot of cluster 2 isoforms after 4 weeks at 40°C, expressed as contours (ranging from 5.5 to 13.68) within the formulation space (with ionic strength fixed at 95 mM NaCl) at various protein concentrations (Vintrofus alpha mg / mL) and pH. This data was obtained by cIEF identification and isoform distribution by iCE3.

[0028] [Figure 10] Figure 10 is a contour plot of the % oxidation level of Met516 after 4 weeks at 40°C, expressed as a contour (range 5.77-8.76) within the formulation space (with ionic strength fixed at 95 mM NaCl) at various protein concentrations (Vintrofus Alpha mg / mL) and pH. This data was obtained by RP-UPLC.

[0029] [Figure 11] Figure 11 is a contour plot of % oxidation of Met516 after 4 weeks at 40°C, expressed as contours (see contour lines at 6, 6.2, 6.4, and 6.6) within the formulation space (with pH fixed at 6.25) at various protein concentrations (Vintrofus Alpha mg / mL) and ionic strengths (mM NaCl). This data was obtained by RP-UPLC.

[0030] [Figure 12] FIG. 12 is a scatter plot for all samples showing % oxidation vs. vine trough alpha concentration after light stress.

[0031] [Figure 13] FIG. 13 is a scatter plot for all samples showing % oxidation versus ionic strength after light stress.

[0032] [Figure 14] FIG. 14 is a scatter plot for all samples showing % oxidation versus pH after light stress.

[0033] [Figure 15] Figure 15 shows 2D and 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) expressed as a contour line (in the 2D plot) and a surface (in the 3D plot) within the formulation space (with a fixed protein concentration of 20 mg / mL) at various pHs and ionic strengths (given as mM NaCl), suggesting that the optimum for IgG:TGFβR2 at this concentration is pH 5.7 and an ionic strength of 40 mM NaCl.

[0034] [Figure 16] Figure 16 shows 2D and 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) expressed as a contour line (in the 2D plot) and a surface (in the 3D plot) within the formulation space (with a fixed protein concentration of 40 mg / mL) at various pHs and ionic strengths (given as mM NaCl), suggesting that the optimum for IgG:TGFβR2 at this concentration is pH 5.9 and an ionic strength of 60 mM NaCl.

[0035] [Figure 17]Figure 17 shows 2D and 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) expressed as a contour line (in the 2D plot) and a surface (in the 3D plot) within the formulation space (with a fixed protein concentration of 60 mg / mL) at various pHs and ionic strengths (given as mM NaCl), suggesting that the optimum for IgG:TGFβR2 at this concentration is pH 5.9 and an ionic strength of 150 mM NaCl.

[0036] [Figure 18] FIG. 18 is a graph showing how % HMW varies with protein concentration after 4 weeks of temperature stress at 40° C. when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measurements indicated by circles in the graph).

[0037] [Figure 19] FIG. 19 is a graph showing how % HMW varies with protein concentration after 8 weeks of temperature stress at 40° C. when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measurements indicated by circles in the graph).

[0038] [Figure 20]FIG. 20 is a graph showing how Main Clipping varies with protein concentration after 4 weeks of temperature stress at 40° C. when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied Excipients" is shown in the legend (the legend refers to predicted values ​​based on measurements indicated by circles in the graph).

[0039] [Figure 21] 21 is a graph showing how %LMW varies with "varied excipients" after 4 weeks of temperature stress at 40° C. when the surfactant is fixed as polysorbate 20 and the ionic strength is fixed at the NaCl concentration applied to a particular protein concentration with polysorbate 20 in Table 3J. Measured values ​​are shown as circles, while predicted values ​​based on measured values ​​are shown as squares.

[0040] [Figure 22] FIG. 22 is a graph showing how % deamidated forms vary with protein concentration after 4 weeks at 40° C. when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0041] [Figure 23] Figure 23 is a graph showing how the % oxidized form varies with excipient after 4 weeks at 40°C when the protein (Vintrafus Alpha) concentration is fixed at 40 mg / mL and the surfactants are fixed at polysorbate 20 (squares represent predicted values) and corifol 188 (triangles represent predicted values ​​based on measured values, represented by circles).

[0042] [Figure 24] FIG. 24 is a graph showing how the % oxidized form varies with excipient after 8 weeks at 40° C. when the protein (Vintrafus alpha) concentration is fixed at 40 mg / mL.

[0043] [Figure 25] Figure 25 is a graph showing how % Cluster 1 (by iCE3) varies with excipient after 4 weeks of temperature stress at 40°C when the protein (Vintrafus alpha) concentration is fixed at 40 mg / mL. Circles represent measured values, and squares represent predicted values ​​based on the measured values.

[0044] [Figure 26] FIG. 26 is a graph showing how % Cluster 2 (iCE3) varies with protein concentration after 4 weeks at 40° C. when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0045] [Figure 27] FIG. 27 is a graph showing how %NMW varies with protein concentration (by SE-UPLC) after light stress when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0046] [Figure 28]FIG. 28 is a graph showing how primary clipping (by CGE) varies with protein concentration after light stress when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measurements indicated by circles in the graph).

[0047] [Figure 29] FIG. 29 is a graph showing how %LMW (by CGE) varies with protein concentration after light stress when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0048] [Figure 30] FIG. 30 is a graph showing how % oxidized (by RO-UPLC) varies with protein concentration after light stress when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0049] [Figure 31] FIG. 31 is a graph showing how % HMW varies with protein concentration after three freeze-thaw cycles when the surfactant is fixed as polysorbate 20 and the NaCl concentration (ionic strength) is that used in Table 3J for a particular protein concentration with polysorbate 20, and "Varied excipients" is shown in the legend (the legend refers to predicted values ​​based on measured values ​​indicated by circles in the graph).

[0050] [Figure 32] FIG. 32 is a bar graph showing the variation of %HMW (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 3J are shown on the horizontal axis) before and after 3 days of mechanical stress (300 rpm).

[0051] [Figure 33] FIG. 33 is a graph showing how % HMW (after mechanical stress) varies with surfactant when all other factors are averaged.

[0052] [Figure 34] FIG. 34 is a graph showing how overall desirability (based on a balance of factors, stress tests, and results) varies by excipient for protein (Vintrafus Alpha) concentrations of 40 mg / mL (predicted values ​​are shown as squares, which are based on measured values ​​shown as circles), 50 mg / mL (predicted values ​​are shown as triangles, which are based on measured values ​​shown as circles), and 60 mg / mL (predicted values ​​are shown as diamonds, which are based on measured values ​​shown as circles).

[0053] [Figure 35] Figure 35 is a graph showing how overall desirability (based on a balance of factors, stress tests, and results) varies with excipient for the surfactant polysorbate 20 (predicted values ​​are shown as squares, which are based on measured values, which are shown as circles) and corifol 188 (predicted values ​​are shown as triangles, which are based on measured values, which are shown as circles) when the protein concentration is fixed at 40 mg / mL.

[0054] [Figure 36] FIG. 36 is a bar graph showing the % HMW increase (by SE-UPLC) after heat stress for each formulation in Table 3K and additional formulation 2A.5 (2A.5) in Table 2A (designated "01-300518").

[0055] [Figure 37] FIG. 37 is a bar graph showing the %LMW increase (by CGE-non-reduced) after heat stress for each formulation in Table 3K and 2A.5 in Additional Table 2A (designated "01-300518").

[0056] [Figure 38] FIG. 38 is a bar graph showing % oxidation (by RP-UPLC) after heat stress for each formulation in Table 3K and 2A.5 (designated "01-300518") in Additional Table 2A.

[0057] [Figure 39] FIG. 39 is a bar graph showing % deamidation (by IEX) after heat stress for each formulation in Table 3K and additional Table 2A, 2A.5 (designated "01-300518").

[0058] [Figure 40] FIG. 40 is a bar graph showing the % purity (by CGE-reduced, i.e., CGE under reducing conditions) after heat stress for each formulation in Table 3K and 2A.5 in Additional Table 2A (designated "01-300518").

[0059] [Figure 41] FIG. 41 is a bar graph showing % primary clipping (by CGE-reduction) after heat stress for each formulation in Table 3K and 2A.5 (designated "01-300518") in Additional Table 2A.

[0060] [Figure 42] FIG. 42 is a bar graph showing the % HMW increase (by SE-UPLC) after light stress for each formulation in Table 3K.

[0061] [Figure 43] FIG. 43 is a bar graph showing % oxidation (by RP-UPLC) after light stress for each formulation in Table 3K.

[0062] [Figure 44] FIG. 44 is a bar graph showing the %LMW (by CGE-non-reduced) after light stress for each formulation in Table 3K.

[0063] [Figure 45] FIG. 45 is a bar graph showing the % purity (by CGE-reduction) after light stress for each formulation in Table 3K.

[0064] [Figure 46] FIG. 46 is a bar graph showing % primary clipping (by CGE-reduction) after light stress for each formulation in Table 3K.

[0065] [Figure 47] FIG. 47 is a bar graph showing the % HMW (by SE-UPLC) after mechanical stress for each formulation in Table 3K.

[0066] [Figure 48] FIG. 48 is a bar graph showing the %LMW (by CGE-non-reduced) after mechanical stress for each formulation in Table 3K.

[0067] [Figure 49] FIG. 49 is a bar graph showing the % purity (by CGE-reduction) after mechanical stress for each formulation in Table 3K.

[0068] [Figure 50] FIG. 50 is a bar graph showing % primary clipping (by CGE-reduction) after mechanical stress for each formulation in Table 3K.

[0069] [Figure 51] FIG. 51 shows a time versus temperature graph illustrating how the 3FT cycle was performed.

[0070] [Figure 52]FIG. 52 is a bar graph showing how pH varies at various time points during stress testing for all of formulations F1-F20, including (bars are listed from left to right for each formulation): time=0; after 3FT 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.

[0071] [Figure 53] FIG. 53 is a bar graph showing the osmolality for each of formulations F1-F20.

[0072] [Figure 54] Figure 54 is a bar graph showing how turbidity varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0073] [Figure 55] Figure 55 is a bar graph showing how the temperature of the Tm2 peak of the nano-DSC traces varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0074] [Figure 56] Figure 56 is a bar graph showing how the onset temperature, Tm, of the nano-DSC traces varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0075] [Figure 57(A)]Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C. [Figure 57(B)] Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C. [Fig. 57(C)] Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C. [Fig. 57(D)] Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C. [Figure 57(E)] Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C. [Fig. 57(F)]Figure 57 is a bar graph showing how %HMW varies (by SE-UPLC) by formulation (formulation numbers corresponding to those in Table 5A are provided on the horizontal axis). (A), (C), and (E) show the %HMW before and after 4 weeks at 40°C, and (B), (D), and (F) show the difference in %HMW between time = 0 and after 4 weeks at 40°C.

[0076] [Figure 58(A)] Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C. [Figure 58(B)] Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C. [Figure 58(C)] Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C. [Figure 58(D)] Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C. [Figure 58(E)]Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C. [Figure 58(F)] Figure 58 is a bar graph showing how %LMW (by CGE-non-reduced) varies by formulation (formulation numbers corresponding to those in Table 5A are provided 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 difference in %LMW between time = 0 and after 4 weeks at 40°C.

[0077] [Figure 59] Figure 59 is a 3D contour plot with the desirability parameter (reflecting the balance of factors in the overall response assessment) represented as a surface in formulation space with varying trehalose concentration and ionic strength (given as mM NaCl) and fixed arginine concentration.

[0078] [Figure 60] Figure 60 is a 3D contour plot with the desirability parameter (reflecting the balance of factors in the overall response assessment) represented as a surface in formulation space with varying arginine concentration and ionic strength (given as mM NaCl) and fixed trehalose concentration.

[0079] [Figure 61] Figure 61 is a 3D contour plot with the desirability parameter (reflecting the balance of factors in the overall response assessment) represented as a surface in formulation space with varying arginine and trehalose concentrations and a fixed concentration of ionic strength (given as mM NaCl). DETAILED DESCRIPTION OF THE INVENTION

[0080] Detailed Description of the Invention definition Unless otherwise specified, the following terms used in the specification and claims have the following meanings indicated below.

[0081] Throughout the description and claims of this specification, the words "comprise" and "include" and variations thereof mean "including but not limited to" and are not intended to exclude (and do not exclude) other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification should be understood to contemplate plural as well as singular presence unless the context otherwise requires.

[0082] As used herein, reference to an "antibody," such as an IgG (e.g., when used in reference to an IgG:TGFβR fusion protein, etc.) preferably refers to a complete antibody or an antigen-binding fragment thereof, where the antigen-binding fragment preferably comprises at least the Fv region or ScFv, and more preferably comprises at least the Fab region or F(ab)2 fragment. However, in more preferred embodiments, reference to an "antibody," such as an IgG (or any subtype thereof, such as IgG1, IgG4, etc.) refers to a complete antibody.

[0083] In its monomeric form, an intact antibody generally comprises two fragment antigen-binding (Fab) regions (or Fab domains), arbitrarily designated F(ab)2, connected, typically via a hinge region, to a fragment crystallizable (Fc) region (or Fc domain). Such a structure is composed of four polypeptide chains—two identical heavy chains and two identical light chains—all interconnected by disulfide bridges. Each Fab domain comprises one heavy chain (e.g., V) linked via disulfide bridges. H -C H 1) and one light chain (e.g., V L -C LThe Fc domain comprises the paired portions of the two heavy chains (e.g., C H 2-C H 3) includes the pair.

[0084] Intact IgG is a monomeric antibody and has the structure previously described for intact antibodies. In the case of intact IgG, each identical heavy chain comprises, in order, starting from the N-terminus and ending at the C-terminus, a variable heavy chain region (V H ), the first constant heavy chain region (C H 1), the second constant heavy chain region (C H 2), and a third constant heavy chain region (C H 3), while each identical light chain contains, in order, a variable light chain region (V L ), and the constant light chain region (C L ) containing the heavy chain V H -C H 1 part and V of the light chain L -C L are cross-linked together via disulfide bridges to form one half of the pair of Fab regions of intact IgG. H 2-C H The three portions are cross-linked together via disulfide bridges to form the Fc region of intact IgG. Intact IgG has a molecular weight of 140 kDa to 180 kDa, preferably either 140 to 160 kDa (more preferably 144 to 155 kDa, most preferably about 146 kDa) or 165 to 175 kDa (more preferably 168 to 172 kDa, most preferably about 170 kDa). Intact IgG typically has a pI of 6 to 9.5.

[0085] There are four distinct subclasses of intact IgG antibodies, designated IgG1, IgG2, IgG3, and IgG4. All subclasses have the same core region as described above, but vary slightly, particularly with respect to the number of amino acids and / or disulfide bonds in the hinge region. IgG1 and IgG4 may be considered the most similar from a structural standpoint. Intact IgG1, IgG2, or IgG4 typically have a molecular weight of 140-160 kDa (more preferably 144-155 kDa, most preferably approximately 146 kDa), whereas intact IgG3 typically has a molecular weight of 165-175 kDa (more preferably 168-172 kDa, most preferably approximately 170 kDa). Intact IgG1 typically has a pI of 8-9.4, more preferably 8.2-9.2. Intact IgG2 typically has a pI of 6.5-8.5, more preferably 7.0-8.0. Typically, intact IgG3 has a pI of 7 to 9.5, more preferably 7.5 to 9.0. Typically, intact IgG4 has a pI of 6 to 8.5, more preferably 6.4 to 8.

[0086] An antigen-binding fragment may include, for example: a Fab region (e.g., a region of its corresponding light chain V L -C L and paired V H -C H a F(ab)2 region consisting of two connected Fab regions; a Fv region (e.g., a corresponding variable light chain portion V L and paired V H and / or a single chain variable region fragment ScFv domain (e.g., a VFv domain connected via a peptide linker, preferably comprising about 25 amino acids). L V connected to H). In the context of the present invention, reference to IgG, for example in IgG:TGFβR fusion protein (including more specifically defined fusion proteins such as anti-PD-L1(IgG):TGFβR2 fusion protein) may refer to, for example, a complete IgG fused to TGFβR2, or an antigen-binding fragment, as described above, for example, fused to TGFβR2. Most preferably, however, complete IgG is present in such fusion proteins.

[0087] The terms "TGF-β receptor" (TGFβR), as well as "TGF-β receptor I" (TGFβR1) or "TGF-β receptor II" (TGFβR2), are well known in the art. For the purposes of this disclosure, reference to such receptors includes the complete receptor and fragments capable of binding TGF-β. Preferably, this is the extracellular domain of the receptor or a fragment of the extracellular domain capable of binding TGF-β.

[0088] The term "fusion protein" is well understood in the art. An IgG:TGFβR fusion protein is an IgG antibody (preferably a monoclonal antibody, preferably in homodimeric form) fused to a TGF-β receptor. The nomenclature anti-PD-L1 (IgG1):TGFβR2 fusion protein refers to an anti-PD-L1 IgG1 antibody fused to TGF-β receptor II, preferably to a fragment of its extracellular domain that is capable of binding TGF-β.

[0089] "Vintraffsalpha" is well understood in the art. Vintraffsalpha is an anti-PD-L1 (IgG1):TGFβR2 fusion protein and is described in CAS Registry Number 1918149-01-5. It is also described in WO2015118175 and further detailed in Lan et al. (Lan et al., "Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β," Sci. Transl. Med. 10, 2018, pp. 1-15). Specifically, Vintraffsalpha is a fully human immunoglobin G1 (IgG1) monoclonal antibody against human PD-L1 fused to the extracellular domain of human TGFβ receptor II (TGFβR2). Thus, Vintraffsalpha is a bifunctional fusion protein that simultaneously blocks the PD-L1 pathway and the TGFβ pathway. In particular, WO2015118175 describes bintrafsalfa on page 34 of Example 1 as follows (bintrafsalfa is referred to in this passage as an “anti-PD-L1 / TGFβ trap”): "Anti-PD-L1 / TGFβ trap is an anti-PD-L1 antibody-TGFβ receptor II fusion protein. The light chain of the molecule is identical to the light chain of the anti-PD-L1 antibody (SEQ ID NO: 1). The heavy chain of the molecule (SEQ ID NO: 3) is a fusion protein comprising the heavy chain of the anti-PD-L1 antibody (SEQ ID NO: 2) genetically fused to the N-terminus of 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 to reduce proteolytic cleavage."

[0090] For purposes of this disclosure and for molar calculations, the molecular weight of vintrofs alpha (M w) is considered to be 182 kilodaltons (kDa), or 182,000 g / mol. Thus, a liquid pharmaceutical composition containing 10 mg / mL Vitrafsalfa can be considered as a 0.055 mM solution, 40 mg / mL as a 0.220 mM solution, and 50 mg / mL as a 0.275 mM solution.

[0091] Reference herein to any particular fusion protein, including any of the IgG:TGFβR fusion proteins described herein (particularly vintrafs alpha), includes the associated drug substance, whether commercially available, described in the patent literature, or otherwise described in the art, as well as biosimilars thereof.

[0092] Reference herein to any particular IgG:TGFβR fusion protein defined by reference to a defined nucleotide or amino acid sequence described herein (particularly IgG:TGFβR2 fusion proteins, and more particularly IgG:TGFβR2 fusion proteins having the amino acid sequence of bintrofus alpha) may include variants that (1) share 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 for that IgG:TGFβR fusion protein, or (2) have no more than 30%, no more than 20%, no more than 10%, or no more than 5% difference in amino acid residues from the disclosed amino acid sequence for that IgG:TGFβR fusion protein. Preferably, reference herein to any particular IgG:TGFβR fusion protein defined by reference to a nucleotide or amino acid sequence described herein (particularly IgG:TGFβR2 fusion proteins, and more particularly IgG:TGFβR2 fusion proteins having the amino acid sequence of bintrofus alpha) refers to a protein having that exact sequence without any variant thereof.

[0093] 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).

[0094] A statement that a protein "differs" in a certain number of amino acid residues means that such number of residues are either inserted, deleted, and / or substituted relative to a reference sequence.

[0095] As used herein, the terms "buffer," "buffer system," or "buffered solution" generally refer to an aqueous solution containing a mixture of an acid (usually a weak acid, e.g., acetic acid, citric acid, or the imidazolium form of histidine) and its conjugate base (e.g., acetate or citrate, e.g., sodium acetate, sodium citrate, or histidine), or alternatively, a base (usually a weak base, e.g., histidine) and its conjugate acid (e.g., a protonated histidine salt). The pH of a "buffered solution" will change only slightly upon the addition of small amounts of strong acid or strong base due to the "buffering effect" provided by the buffer system. A buffered solution may contain more than one buffer system, but preferably only two (i.e., dual-buffer systems such as histidine-citrate, histidine-acetate, and citrate-phosphate), but most preferably the buffered solution contains one and at most one buffer system.

[0096] In the present context, a "strong acid" is preferably an acid with a pK of -1.0 or less. a whereas a "weak acid" is one having a pK of preferably 2.0 or greater. aIn the context of this specification, a "strong base" is preferably one whose conjugate acid has a pK of 12 or more (preferably 14 or more). a whereas a "weak base" is one whose conjugate acid preferably has a pK of 10 or less. a It has the following characteristics.

[0097] Unless otherwise stated, the term "pK a " refers to the pK in water at standard ambient temperature and pressure (SATP) of the conjugate acid of the relevant species. a It should be interpreted as a value.

[0098] An "amino acid component" is a component or components that contain one or more amino acids, although an amino acid component may consist of a single amino acid.

[0099] Unless otherwise stated, reference herein to an "amino acid" or "amino acids," whether specific (e.g., arginine, histidine) or general (e.g., any amino acid), refers to their presence or otherwise in the context of a composition (particularly a pharmaceutical liquid composition of the present invention). Free amino acid(s) (regardless of its / their protonation state and / or salt form, for consistency the amount is preferably calculated with reference to the free amino acids themselves). This may preferably include natural and / or artificial amino acids. Unless stated to the contrary, such references are not to be construed as limiting the scope of the present invention as they relate to the free amino acids of larger compounds such as peptides or proteins (wherein such amino acid residues are linked via peptide bonds). As part covalently incorporated Amino acid residue(s) The term "antibody" is not intended to refer to a compound (as opposed to a composition containing multiple compounds). Thus, antibodies as proteins contain amino acid residues, but are not considered to contain any "free amino acid(s)." By way of example, a composition defined as being "arginine-free" does not contain any free arginine, although it may still contain one or more proteins (e.g., vintrafs alpha) that themselves contain arginine residues.

[0100] Unless otherwise stated, any reference herein to one or more "amino acids", whether specifically or generally, preferably refers to the L-stereoisomers or their racemates, most preferably to L-amino acids.

[0101] As used herein, a "sugar component" is a component or components that comprise one or more sugar(s) and / or sugar alcohol(s), although a sugar component can consist of a single sugar or sugar alcohol.

[0102] As used herein, a "non-reducing sugar" is a sugar that does not have an aldehyde moiety or the ability to form an aldehyde moiety (eg, through isomerization).

[0103] As used herein, "tonicity modifying agent" or "tonicity agent" refers to an agent whose inclusion within a composition contributes to (or increases) the overall osmolality and osmolality of the composition. Preferably, a tonicity agent as used herein includes a substance that functions to make a solution resemble the osmotic characteristics of body fluids.

[0104] As used herein, an "antioxidant" or "antioxidant component" refers to a component or components that contain one or more antioxidant compounds, although an antioxidant component may consist of a single antioxidant compound. Preferably, the antioxidant, in the context of the compositions of the present invention, reduces oxidation of groups within the fusion protein that would otherwise be susceptible to oxidation.

[0105] "Chelating agent" is a term of art that refers to a compound that is capable of complexing, preferably in a multidentate manner, with various groups, molecules, atoms, or ions, and may itself exert antioxidant activity.

[0106] As used herein, when a composition is said to be "characterized by the absence of [a particular component]," this means that the composition in question is either substantially free or completely free of that component.

[0107] The term "substantially free," when used in reference to a given component of a composition (e.g., a "liquid pharmaceutical composition substantially free of amino acid components"), refers to a composition to which essentially no amino acid component has been added. As explained above, such reference does not relate to the presence of the amino acid residue(s) within the protein structure. When a composition is "substantially free" of a given component, the composition preferably contains less than 0.1 wt% of the component, preferably less than 0.01 wt%, preferably less than 0.001 wt%, preferably less than 0.0001 wt%, preferably less than 0.00001 wt%, preferably less than 0.000001 wt%, preferably less than 0.000001 wt%, and most preferably less than 0.0001 ppb (parts per billion by weight).

[0108] The term "completely free," when used in reference to a given component of a composition (e.g., "a liquid pharmaceutical composition completely free of amino acid components"), refers to a composition that is free of that component. As explained above, such references with respect to amino acids present in a pharmaceutical composition do not relate to the presence of the amino acid residue(s) within a protein structure.

[0109] Preferably, unless otherwise stated, parameters that can be affected by pressure and / or temperature (e.g., pH, pK a When reference is made to a temperature (e.g., a liquid, a gas, etc.) or state of matter (e.g., a liquid, a gas, etc.), preferably, in the absence of further clarification, such reference refers to that parameter at standard ambient temperature and pressure (SATP), which is a temperature of 298.15 K (25°C, 77°F) and a pressure of 100 kPa absolute (14.504 psi, 0.987 atm).

[0110] Reference herein to a specific amount of a given component of a composition, particularly a buffer or buffer system, surfactant, sugar component, amino acid component, tonicity agent, antioxidant, and / or chelating agent, preferably refers to the amount of the pure anhydrous form of the relevant component (or a composition formed by using that amount of the pure anhydrous form), even if such component is used in hydrous form when forming the composition. The amount of any corresponding hydrous form (e.g., monohydrate, dihydrate, etc.) can be readily calculated by simply using the appropriate multiplication. For example, unless otherwise stated (such as in the examples where the amounts relate to trehalose dihydrate), a specified amount related to trehalose refers to the anhydrous form of trehalose (or to a composition formed by using the specified amount / concentration of anhydrous trehalose), which has a molecular weight of 342.296 g / mol. Therefore, to calculate the corresponding amount of trehalose dihydrate needed to form the same composition (less water would be added), it is necessary to multiply the specified amount by 378.33 / 342.296, since the molecular weight of trehalose dihydrate is 378.33. Those skilled in the art will readily understand how to carefully adjust the amount of diluent / water depending on the type of ingredient used to arrive at the target concentration. Obviously, this issue does not apply when molar amounts are specified.

[0111] As used herein, the term "pharmaceutical composition" refers to a formulation that is suitable for mammalian treatment, e.g., in the sense that it does not contain overly toxic components. References herein to a "composition" generally refer to a pharmaceutical composition as defined herein.

[0112] As used herein, the term "stable" generally refers to the physical and / or chemical and / or biological stability of an ingredient, typically its activity or composition, during storage / preservation. With respect to aqueous compositions of biologics, storage stability preferably means that the biologic is sufficiently stable (i.e., with the prescribed limitations for patient safety) when stored at 2-8°C for at least 6 months, preferably at least 12 months, and preferably up to 24 months. However, accelerated stability studies may be used to provide relevant stability information.

[0113] References to "treating" or "treatment" should be understood to include prevention as well as alleviation of established symptoms of a condition. Accordingly, "treating" or "treatment" of a condition, disorder, or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder, or condition from developing in a person afflicted with or predisposed to the condition, disorder, or condition but who has not yet experienced or exhibited clinical or preclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the development of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or preclinical symptom thereof; or (3) eliminating or alleviating the disease, i.e., causing regression of the condition, disorder, or condition or at least one clinical or preclinical symptom thereof.

[0114] In the context of this invention, a "therapeutically effective amount" or "effective amount" of a pharmaceutical composition means an amount that is effective when administered to a mammal for treating a disease or disorder, in both prophylactic and therapeutic aspects, and the pharmaceutical composition is effective in treating the disease of concern. A "therapeutically effective amount" will vary depending on the fusion protein, the disease and its severity, and the age, weight, etc. of the mammal being treated.

[0115] Amounts specified herein for ingredients and components, whether specified in terms of "parts," ppm (parts per million), percentages (% e.g., wt%), or ratios, are intended to be by weight unless otherwise stated.

[0116] 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 percentage of that component by weight relative to the total weight of the composition as a whole. It will be understood by those skilled in the art that the sum of the weight percentages of all components of a composition (whether specified or not) totals 100 wt%. However, if not all components are listed (e.g., the composition is said to "contain" one or more specified components), the balance of the weight percentage may optionally be made up to 100 wt% by unspecified components (e.g., a diluent such as water, or other nonessential but suitable additives).

[0117] As used herein, the amount or concentration of a particular component may be provided as a weight / volume percentage, e.g., expressed as a number between 0 and 100 followed by "% w / v," "%(w / v)," "w / v%," "wt / vol%," or "% wt / vol," particularly when the component is present in a liquid composition (preferably an aqueous solution). It will be readily understood by those skilled in the art that a given % w / v may be converted to other weight / volume units, such as "mg / mL." A component said to be present at 1% (w / v) is present at a concentration of 10 mg / mL (i.e., the mg / mL number is the number given in % w / v multiplied by 10). It will also be understood by those skilled in the art that the density of the overall composition is approximately 1 g / cm. 3 It will be understood that where appropriate, % w / v (particularly % w / v ranges) may be re-expressed as wt % (i.e., % w / w) (particularly wt % ranges). This is appropriate in the case of pharmaceutical compositions of the present invention. Weight ratios between components may therefore be devised from the information provided in this application.

[0118] When a composition is said to contain multiple specified components (optionally in specified amounts of concentrations), the composition may optionally include additional components other than those specified. However, in some embodiments, a composition said to contain multiple specified components, in any specified amount, can actually consist essentially of or consist of all of the specified components. In any situation, the individual components can contain, consist essentially of, or consist of themselves, a subcomponent, or one or more subcomponents. In this specification, whenever the term "containing" is used, it can be replaced by "consisting essentially of" or "consisting of," where appropriate.

[0119] As used herein, when a composition is referred to as "consisting essentially of" a particular component or components, the composition preferably contains at least 70 wt% of the component(s), preferably at least 90 wt% of it, preferably at least 95 wt% of it, and most preferably at least 99 wt% of it. Preferably, a composition referred to as "consisting essentially of" a particular component consists of that component except for one or more trace impurities.

[0120] "About," when used to modify a parameter (e.g., pH value) defined by a number, means that the parameter may vary, for example, within the experimental precision of determining the parameter, or by an amount that is 5% below or above the numerical value stated for that parameter, and preferably by an amount that is 2% below or above the numerical value stated for that parameter. In a preferred embodiment, a parameter described by the term "about" corresponds to the numerical value stated.

[0121] In this specification, whenever a component is specified as being capable of ionization (e.g., protonation or deprotonation), unless incompatible in a given context, the definition of the component preferably includes the definition of any suitable salt thereof, preferably a pharmaceutically acceptable salt thereof. For example, this applies to any reference herein to buffer substances (e.g., citric acid or citrate salts), amino acids, and the like. Similarly, whenever a component is specified as being capable of neutralization, unless incompatible in a given context, the definition of the component preferably includes their neutralized form—e.g., citrate salts instead of citric acid.

[0122] "Isoelectric point" (pI) refers to the pH at which a given molecule (or portion thereof) is, from statistical prediction, electrically neutral - i.e., has no net charge. pI is of particular relevance to proteins, including fusion proteins of the invention, because they contain functional groups that can be positive, negative, neutral, and polar, depending on the prevailing pH of the local environment. The pI of any given molecule or portion thereof may be determined experimentally by methods well known in the art. However, pI may also be calculated using a variety of methods well known in the art. Preferably, pI is determined experimentally.

[0123] General points and advantages of the invention It is generally known that when developing a viable antibody formulation, and in particular an antibody-fusion protein formulation, various factors affect the stability of the formulation. In the case of the present invention, the fusion of the antibody with the receptor further complicates formulation development, as both portions of the molecule must comply with requirements.

[0124] This invention arose following a skillful and intuitive targeting and careful exploration of the generally unproductive formulation space to identify which excipients are important and their relative amounts must be present or absent to best complement the associated fusion protein during processing and / or storage. Without such a well-targeted and careful approach, formulation scientists are unlikely to design a viable formulation.

[0125] The many advantages of the present invention, as well as the challenges actually associated with its conception and development, will be self-evident. The inventive effort set forth in this disclosure represents a significant contribution to the art, and despite the aforementioned unpredictability, the invention, given its reinforcement by the examples and data presented herein, establishes a viable and reasonable formulation space, a contribution that is commensurate with the scope of the present invention.

[0126] Pharmaceutical Composition The present invention provides a pharmaceutical composition. Preferably, the pharmaceutical composition is a liquid pharmaceutical composition, preferably an aqueous pharmaceutical composition (thus comprising water, preferably water for injection, as a diluent). However, the pharmaceutical composition may also be a lyophilized composition (i.e., a lyophilized pharmaceutical composition, preferably a solid). Such a lyophilized composition may preferably be reconstituted to provide a liquid pharmaceutical composition, preferably an aqueous pharmaceutical composition. The definitions and amounts provided herein may relate to either or both the liquid composition and / or the lyophilized composition. When an amount is specified in terms of a concentration within a liquid composition (e.g., as weight %, weight / volume, or either volume molality or weight molality), this may be converted to a concentration ratio (either weight ratio or molar ratio) in a solid composition by simple calculations known in the art, for example, using knowledge of the molecular weights of the relevant components to enable conversion between the two common units (e.g., parts or weight %, or moles).

[0127] The pharmaceutical composition contains an IgG:TGFβR fusion protein and, optionally, one or more pharmaceutically acceptable excipients and / or carriers. Preferably, the pharmaceutical composition contains one or more of the following: a buffer system, a surfactant, a sugar component, an amino acid component, a tonicity agent, an antioxidant, and a chelating agent. Liquid pharmaceutical compositions (either formulated per se or in the reconstituted form of a lyophilized formulation) preferably contain a diluent, such as water (e.g., water for injection). Preferably, the pharmaceutical composition is characterized by a pH of 4 to 8. Particularly when the pharmaceutical composition is intended for subcutaneous injection without prior dilution, the pharmaceutical composition is preferably characterized by an osmolality of 240 to 640 mOsm / kg.

[0128] It will be understood that a compound of the pharmaceutical composition can fulfill more than one role. For example, histidine, an amino acid component, may also function as part of a buffer system. Thus, when a pharmaceutical composition containing a buffer system and an amino acid component is referred to, such an embodiment encompasses a pharmaceutical composition with a histidine buffer system and no additional amino acid components. Thus, unless incompatible in a given context, for example, when an embodiment refers to non-overlapping concentration ranges for each feature of the pharmaceutical composition, the recited properties of the pharmaceutical composition may be fulfilled by one or more compounds (i.e., multifunctional compounds) that fulfill two or more of the recited properties. In some embodiments, the recited properties of the pharmaceutical composition are fulfilled by individual compounds. In some embodiments, the recited properties of the pharmaceutical composition are fulfilled by one or more compounds that fulfill two or more of the recited properties.

[0129] In certain embodiments, the pharmaceutical composition contains and / or is characterized by one or more of the following (or, in the case of an aqueous composition, consists of one or more of the following, optionally together with water for injection): an IgG:TGFβR fusion protein; a buffer system; a pH of 4-8; a surfactant; a sugar component; an amino acid component; a tonicity agent; an antioxidant; and / or a chelating agent.

[0130] The following numbered paragraphs A1 through A9 disclose specific embodiments of the present invention. A1. A pharmaceutical composition containing an IgG:TGFβR fusion protein. A2. The pharmaceutical composition of A1, further comprising an isotonicity agent. A3. The pharmaceutical composition of A1 or A2, further characterized by a pH of 4 to 8. A4. Any of the pharmaceutical compositions of A1-A3, further comprising a buffer system. A5. Any of the pharmaceutical compositions of A1-A4, further comprising a surfactant. A6. Any of the pharmaceutical compositions of A1-A5, further comprising a sugar component. A7. Any of the pharmaceutical compositions of A1-A6, further containing an antioxidant. A8. Any of the pharmaceutical compositions of A1-A7, further comprising an amino acid component. A9. Any of the pharmaceutical compositions of A1-A8, further comprising a chelating agent.

[0131] Fusion proteins The pharmaceutical composition contains an IgG:TGFβR fusion protein.

[0132] The IgG:TGFβR fusion protein is preferably an IgG:TGFβR fusion protein comprising an IgG fused to the soluble extracellular domain of TGFβR, more preferably the soluble extracellular domain of TGFβR2, or a fragment thereof capable of binding to TGF-β.

[0133] This IgG:TGFβR fusion protein is preferably an IgG:TGFβR fusion protein in which the IgG has a pI of 8 to 10, preferably a pI of 8.5 to 9.5, while the TGFβR has a pI of 4.5 to 6, preferably a pI of 4.6 to 5.4.

[0134] The IgG of this IgG:TGFβR fusion protein is preferably selected from the group consisting of anti-PD-L1(IgG) and anti-PD-1(IgG), more preferably it is 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 having the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2), and SEQ ID NO:21 (CDR3), and three light chain CDRs having the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2), and SEQ ID NO:24 (CDR3); (2) three heavy chain CDRs having the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3), and and (3) anti-PD-L1 (IgG) comprising three heavy chain CDRs having the amino acid sequences of SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2), and SEQ ID NO:29 (CDR3), and three light chain CDRs having the amino acid sequences of SEQ ID NO:30 (CDR1), SEQ ID NO:31 (CDR2), and SEQ ID NO:32 (CDR3). In preferred embodiments, the anti-PD-L1 (IgG) comprises heavy chain CDRs having the amino acid sequences of SEQ ID NOs:1, 2, and 3, and light chain CDRs having the amino acid sequences of SEQ ID NOs: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 and heavy chain sequences 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 it is IgG1 or IgG4, and most preferably it is IgG1.

[0135] Preferably, the C of the IgG:TGFβR fusion proteinH 3 domains, C of vintrafs alpha H The amino acid sequence of the three domains of the IgG:TGFβR fusion protein has 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, or at least 96% sequence identity. H The three domains have amino acid sequences that differ from the CH3 domain of bintrofus alpha by no more than 10, no more than 5, or no more than 4 amino acid residues.

[0136] Preferably, the C of the IgG:TGFβR fusion protein H 1 domain is C of bintrafs alpha H The amino acid sequence of the C domain of the IgG:TGFβR fusion protein has 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or at least 91% sequence identity. H 1 domain is C of bintrafs alpha H It has one domain and an amino acid sequence with no more than 20, no more than 10, or no more than 7 amino acid residue differences.

[0137] Preferably, the C of the IgG:TGFβR fusion protein H 2 domain, C of bintrafs alpha H The amino acid sequence of the C domain of the IgG:TGFβR fusion protein has 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or at least 91% sequence identity. H 2 domain, C of bintrafs alpha H It has two domains and amino acid sequences with differences of 20 or less, 10 or less, or 8 or less amino acid residues.

[0138] Variability in the variable domains (light and heavy chains) and in the light chain in general is tolerated.

[0139] The TGFβR of this IgG:TGFβR fusion protein is preferably TGFβR1 or TGFβR2, more preferably TGFβR2. In a preferred embodiment, it is an IgG:TGFβR2 fusion protein, where the IgG has a pI of 8.5-9.5, while the TGFβR2 has a pI of 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, the TGFβR2 is the soluble extracellular domain of TGFβR2, or a fragment thereof capable of binding to TGF-β. Preferably, the TGFβR2 lacks the cytoplasmic domain of TGFβR2. In some embodiments, the TGFβR2 corresponds to the wild-type human TGFβ receptor type 2 isoform A sequence (e.g., the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 (SEQ ID NO:9)) or the wild-type human TGFβ receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI Reference Sequence Accession No. NP_003233 (SEQ ID NO:10)). Preferably, the TGFβR2 comprises or consists of a sequence corresponding to SEQ ID NO:11 or a fragment thereof that is capable of binding to TGFβ. For example, the TGFβR2 may correspond to the full-length sequence of SEQ ID NO:11. Alternatively, it may have an N-terminal deletion. For example, amino acids 1-26 from the N-terminus of SEQ ID NO:11 may be deleted, such as the most N-terminal amino acids 14-21 or 14-26. In some embodiments, the N-terminal 14, 19, or 21 amino acids of SEQ ID NO:11 are deleted. Preferably, TGFβR2 comprises or consists of a sequence 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% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to 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, TGFβR2 has an amino acid sequence that differs from SEQ ID NO: 11 by no more than 25 amino acids.

[0140] Preferably, the TGFβR of the IgG:TGFβR fusion protein has 92% or more sequence identity, 95% or more sequence identity, 99% or more sequence identity, or 100% sequence identity with the amino acid sequence of the TGFβR of the vin-trough alpha. Preferably, the TGFβR of the IgG:TGFβR fusion protein has an amino acid sequence that differs from the TGFβR of the vin-trough alpha by 50 or less, 40 or less, or 25 or less amino acid residues. The TGFβR of the IgG:TGFβR fusion protein preferably has 100 to 160 amino acid residues, more preferably 110 to 140 amino acid residues. In some embodiments, the amino acid sequence of the TGFβR is selected from the group consisting of a sequence corresponding to positions 1-136 of the TGFβR of the vin-trough alpha, a sequence corresponding to positions 20-136 of the TGFβR of the vin-trough alpha, and a sequence corresponding to positions 22-136 of the TGFβR of the vin-trough alpha.

[0141] Preferably, the TGFβR of the IgG:TGFβR fusion protein has 98% or more sequence identity with the amino acid sequence of the TGFβR of the IgG:TGFβR fusion protein, and the C H 3 domains, C of vintrafs alpha H The amino acid sequence of the three domains of the IgG:TGFβR fusion protein has 92% or more sequence identity. Preferably, the TGFβR of the IgG:TGFβR fusion protein has 25 or fewer amino acid residue differences with the amino acid sequence of the TGFβR of the alpha-TGFβR, and the C of the IgG:TGFβR fusion protein has 92% or more sequence identity with the amino acid sequence of the three domains of the IgG:TGFβR fusion protein. H 3 domains, C of vintrafs alpha H The amino acid sequences of the three domains differ by no more than four amino acid residues.

[0142] Preferably, the IgG:TGFβR fusion protein comprises a linker between the IgG and the TGFβR, the linker preferably comprising 5 to 50 amino acid residues, 10 to 30 amino acid residues, or 20 to 27 amino acid residues. Preferably, such a linker comprises at most two different types of amino acid residues. Preferably, the linker comprises glycine amino acid residues and / or serine amino acid residues. Preferably, such a linker has a structure of the formula (Gly x Ser) y Gly, where 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. Most preferably, the linker is of the formula (Gly x Ser) y Gly, where x is 4 and y is either 4 or 5.

[0143] The IgG:TGFβR fusion protein is preferably an IgG:TGFβR2 fusion protein comprising TGFβR2 fused at its N-terminus to the C-terminus of an IgG antibody, optionally via a linker.

[0144] 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 and heavy chains of SEQ ID NO:1 and SEQ ID NO:3, respectively, of WO2015 / 118175. 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 thereof.

[0145] Preferably, the light and heavy chain sequences 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 bintrofs alpha. Most preferably, the IgG:TGFβR fusion protein is bintrofs alpha.

[0146] In certain embodiments, the IgG:TGFβR fusion protein is characterized by the following: · TGFβR with 95% or more sequence identity with the amino acid sequence of TGFβR from Vintrafus alpha; Bint Rahus Alpha C H C, which has over 92% sequence identity with the amino acid sequence of the three domains H 3 domains; Bint Rahus Alpha C H C, which has over 90% sequence identity with the amino acid sequence of one domain H 1 domain; and Bint Rahus Alpha C H C, which has over 90% sequence identity with the amino acid sequence of the two domains H 2 domains.

[0147] In certain embodiments, the IgG:TGFβR fusion protein is characterized by the following: · TGFβR with differences of 25 or fewer amino acid residues from the amino acid sequence of TGFβR of vintrafus alpha; Bint Rahus Alpha C H C with differences of 4 or fewer amino acid residues from the amino acid sequence of the 3 domains H 3 domains; Bint Rahus Alpha C H C with a difference of 7 or fewer amino acid residues from the amino acid sequence of one domain H 1 domain; and Bint Rahus Alpha C HC, which has differences of 8 or fewer amino acid residues between the amino acid sequences of the two domains H 2 domains.

[0148] For example, C of Bintraffic Alpha H 1. C H 2, and / or C H For region-specific comparison of the amino acid sequences of the three domains (and optionally the TGFβR domain as well), the specific sequence assignments of particular regions shown in Figure 1A or Figure 1B may be used. In some embodiments, for example, the C of vintrofs alpha H 1. C H 2, and / or C H For the purposes of region-specific comparison of the amino acid sequences of the three domains (and optionally also the TGFβR domain), the specific sequence assignments of particular regions shown in FIG. 1B may be used.

[0149] Bint Rahus Alpha C H For region-specific comparison of the amino acid sequences of the two domains, see C shown in FIG. 1A or FIG. 1B. H Sequences assigned to the two domains may be used. In some embodiments, the bintrofs alpha C H For region-specific comparison of the amino acid sequences of the two domains, see C shown in Figure 1B. H Sequences assigned to the two domains may be used.

[0150] Notwithstanding the assignment of specific sequences to specific regions shown in Figures 1A and 1B, any reference to either of these figures based on the common assignment of these regions to antibody sequences, e.g., C of vintrafs alpha, H 1. C H 2, and / or C H Region-specific comparisons may be made of the amino acid sequences of the three domains (and optionally the TGFβR domain as well).

[0151] The pharmaceutical compositions preferably contain an amount of IgG:TGFβR fusion protein (e.g., anti-PD-L1(IgG):TGFβR2 or IgG:TGFβR with a bintrofsalpha sequence) 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 compositions contain 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 the anti-PD-L1(IgG):TGFβR2 fusion protein.

[0152] Diluent The pharmaceutical composition preferably contains a diluent. The composition may contain one or more pharmaceutically acceptable diluents, or a mixture thereof. However, most preferably, the composition is an aqueous composition. Most preferably, the diluent is water, and preferably only water. The water is preferably water for injection (WFI).

[0153] Preferably, the diluent can make up the balance of the components in any composition, e.g., the weight percentages of all components total 100%. Preferably, any concentration provided herein with respect to any component of the composition represents the concentration of that component in the diluent mixed with any other components (and preferably dissolved in the diluent).

[0154] The compositions of the present invention are preferably solutions, and are preferably (substantially or completely) free of particles or precipitates.

[0155] However, in embodiments, the pharmaceutical compositions either contain no water or contain at most 10 wt% water, preferably at most 5 wt% water, preferably at most 2 wt% water, preferably at most 1 wt% water. Such embodiments may be, for example, solid pharmaceutical compositions or lyophilized pharmaceutical compositions that can be reconstituted (preferably by addition of water and / or other relevant diluents, e.g., intravenous fluid or saline) prior to use, administration, or (preferably short-term) storage. Such lyophilized formulations may be reconstituted to provide aqueous pharmaceutical compositions disclosed herein (e.g., with components present or absent at concentrations specified herein).

[0156] Buffer system The pharmaceutical composition preferably contains a buffer system. When small amounts of (strong) acid or (strong) base are added to the pharmaceutical composition (or generated therein, potentially as a result of degradation of one or more components of the pharmaceutical composition, most likely the fusion protein), the buffer system preferably acts as a pH buffer to inertia pH changes. Thus, the buffer system preferably maintains a substantially constant composition pH over time, preferably maintaining mitigation against pH-triggered degradation pathways. Preferably, the pharmaceutical composition is sufficiently buffered to resist a pH change of 1 or more pH units upon storage at 2-8°C for 6 months, and preferably to resist a pH change of 0.5 or more pH units, and most preferably to resist a pH change of 0.2 or more pH units under the same storage conditions.

[0157] Preferably, the composition is a buffered solution stabilized by a buffering agent, which is combined with the buffer's conjugate acid or conjugate base, respectively, depending on whether the buffering agent is itself a base or an acid, and whose pH is weakly acidic or weakly basic. Collectively, the buffering agent and its acid / base conjugate may be considered a "buffer system," and in some embodiments, when two or more buffer systems are present, the composition may contain multiple different buffering agents and corresponding acid / base conjugates. Thus, the composition preferably contains a "buffer system" (preferably including buffer(s) and their acid / base conjugate(s)), and any concentrations specified as relating to a buffer system generally refer to the combined concentrations of the buffer(s) and any of their acid / base conjugate(s).

[0158] The pharmaceutical composition preferably contains a buffer system comprising one or more buffer systems. For example, the buffer system can be a dual-buffer system (e.g., histidine-acetate, phosphate-citrate). The pharmaceutical composition most preferably contains a buffer system comprising 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 mentioned herein in connection with buffer systems). The pharmaceutical composition may be sufficiently stable without a buffer system, for example, if the fusion protein provides sufficient self-buffering, which preferably occurs at relatively high concentrations of the fusion protein.

[0159] In some embodiments, the buffer system is selected from a monobasic buffer system (e.g., acetate buffer), a polybasic buffer system (e.g., phosphate buffer), and an amphoteric buffer system (e.g., amino acid buffer, e.g., histidine), an inorganic buffer system (e.g., ammonium buffer, bicarbonate buffer, carbonate buffer, borate buffer, phosphate buffer), an organic buffer system (e.g., carboxylate buffer, organic ammonium buffer, alkanolammonium buffer, zwitterion buffer, amino acid buffer, aromatic nitrogen buffer, sugar buffer), and any combination thereof.

[0160] In other embodiments, the buffer system is a monocarboxylic acid buffer system (e.g., acetate buffer, formic acid buffer, lactate buffer, salicylate buffer, benzoic acid buffer), a dicarboxylic acid buffer system (e.g., succinate buffer, maleic acid buffer, malic acid buffer, fumaric acid buffer, tartrate buffer, adipic acid buffer, hexanedioic acid buffer), a tricarboxylic acid buffer system (e.g., citrate buffer), a zwitterionic buffer system (e.g., amino acid buffer, sulfonic acid zwitterionic buffer, e.g., N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, 2-aminoethanesulfonic acid (AES) buffer, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, 3-(cyclohexylamino)-propanesulfonic acid (CAPS)), an amino acid buffer system (e.g., ammonium hydroxide, ammonium hydroxide, ammonium hydroxide, ammonium hydroxide), an amino acid buffer system (e.g. ... The buffer solution is selected from 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, N,N-bis(2-hydroxyethyl)-glycine (bicine) buffer, N-(2-acetamido)-iminodiacetic acid (ADA) buffer), aromatic nitrogen buffer systems (e.g., imidazole, pyridine buffer), and alkanolammonium 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 (bis-trispropane) buffer).

[0161] 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 containing a single buffer selected from the group consisting of histidine buffer, phosphate buffer, succinate buffer, and citrate buffer.

[0162] The pharmaceutical composition may contain 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 about a 10 mM buffer system.

[0163] The pharmaceutical composition may contain a buffer system to fusion protein molar ratio of 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.

[0164] Most preferably, the buffer system is or comprises a histidine buffer system. For example, the buffer system is or comprises a 5-60 mM histidine buffer system, a 5-15 mM histidine buffer system, or preferably, about a 10 mM histidine buffer system. In other embodiments, the composition contains a histidine buffer system at a molar ratio of histidine buffer system to fusion protein of 1280:1 to 3:1 or 370:1 to 9:1.

[0165] 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 contains a phosphate buffer system at a molar ratio of phosphate buffer system to fusion protein of 1280:1 to 3:1 or 370:1 to 9:1.

[0166] Preferably, the buffer system is or comprises a succinate buffer system. For example, the buffer system is or comprises a 5-60 mM succinate buffer system or a 5-15 mM succinate buffer system. In other embodiments, the composition contains a succinate buffer system at a molar ratio of succinate buffer system to fusion protein of 1280:1 to 3:1 or 370:1 to 9:1.

[0167] Preferably, the buffer system is or comprises a citrate buffer system. For example, the buffer system is or comprises 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 at a molar ratio of citrate buffer system to fusion protein of 1280:1 to 3:1 or 370:1 to 9:1.

[0168] Preferably, the buffer system does not include acetate buffers.

[0169] Unless only one buffer system is specified, the amounts and concentrations for this buffer system refer to the sum of the amounts and concentrations of all buffer systems. The concentration of the buffer system or any particular buffer system can be substituted by a molar ratio.

[0170] pH The pharmaceutical composition preferably has a pH of 4 to 9. Although the fusion protein and any excipients within the pharmaceutical composition are tolerant and remain fairly stable over a range of pH values, certain pHs are particularly advantageous, especially in the presence of certain excipients or combinations thereof.

[0171] In some embodiments, the pharmaceutical composition has a pH of 4 to 8, pH 4.8 to 7.8, pH 5 to 7, pH 4.9 to 6.8, pH 4.5 to 6.0, pH 4.8 to 6.5, pH 5 to 5.4, pH 5.2 to 6.2, pH 5.3 to 6.3, pH 5.2 to 5.8, pH 5.6 to 5.8, pH 5.9 to 6.1, pH 5.2 to 6.2, pH 5.4 to 6.0, or preferably pH 5.4 to 5.6 or pH 5.8 to 6.0. In some embodiments, the pH of the pharmaceutical composition is about 5.5 or about 5.9.

[0172] One of the challenges encountered when formulating IgG:TGFβR fusion protein compositions has been the significant difference between the pI of the IgG portion (typically about 8-10, 8.5-9.5, or about 9.1) and the pI of the TGFβR portion (typically about 4.5-6, 4.6-5.4, or about 4.9). Generally, it is desirable to formulate biologics at a pH greater than 1-2 units from the pI of the biologic or the relevant portion of the biologic. Optimizing pH for bifunctional biologics, whose functional moieties perform at dramatically different pIs, is particularly challenging. In the case of IgG:TGFβR fusion proteins, the TGFβR portion is more impactful in terms of formulation considerations and potentially less stable than the IgG portion. The IgG portion of Vintrafus Alpha, in particular, has been found to be quite resistant to stress conditions and stable for extended periods, even in liquid / non-lyophilized form. In contrast, when the IgG portion of vintrafus alpha was fused to the TGFβR portion, the molecule was found to precipitate more readily and form particles, and phase separation was observed upon formulation, suggesting that the TGFβR portion is less stable than the IgG portion or at least acts destabilizingly in combination with the IgG portion. This reinforces the need for a pH that is particularly far from the pI of the TGFβR portion, e.g., outside the pH range of 3.9 to 5.9, of IgG:TGFβR fusion protein compositions. Surprisingly, pharmaceutical compositions with a pH close to the pI of the TGFβR portion of the IgG:TGFβR fusion protein were nonetheless found to be highly stable.

[0173] With this in mind, the pharmaceutical composition preferably has a pH that is within 2 pH units, more preferably within 1 pH unit, of the pI of the TGFβR portion of the IgG:TGFβR fusion protein (i.e., the difference between the pH of the composition and the pI of the TGFβR portion is less than 2 units or 1 unit, respectively). The pharmaceutical composition preferably has a pH that differs from the pI of the IgG portion of the fusion protein by 2 or more pH units, more preferably by 3 or more pH units. For example, the composition may have a pH of 5.4-6.0, where the pI of the TGFβR portion is 4.4-5.0 and the pI of the IgG portion is 8.4-9.5.

[0174] The present invention encompasses the use or absence of any buffer system as defined herein, but as long as the pH is as defined herein, a histidine buffer system is the most preferred buffer system, most preferably at a pH of 5.2 to 6.2.

[0175] surfactants The pharmaceutical composition preferably contains a surfactant. The pharmaceutical composition preferably contains at most one surfactant. In the context of the present invention, a surfactant can inhibit one or more degradation pathways of the fusion protein or its constituent parts, such as unfolding (and resulting aggregation), aggregation, and sometimes further fragmentation. The surfactant may promote dissolution of the fusion protein. However, the pharmaceutical composition may be characterized by the absence of a surfactant (or any one or more of the surfactants specifically mentioned herein in connection with surfactants).

[0176] Preferably, the surfactant is a non-ionic surfactant, for example, selected from the group consisting of fatty alcohols, fatty alcohol ethers, fatty acid esters, fatty acid amides, polyoxyalkylene alkyl ethers, polyoxyethylene alkyl ethers, non-ionic block copolymers, α-tocopherol, and any combination thereof, or selected from the group consisting of sorbitan esters (Span), ethoxylated sorbitan esters (polysorbates), and block alkoxylates.

[0177] In some embodiments, the surfactant is an ethoxylated fatty acid ester surfactant. In another embodiment, the surfactant is a surfactant selected from the group consisting of polysorbate(s), poloxamer(s), and corrifol(s). Preferably, it is a corrifol surfactant or a polysorbate surfactant. Preferably, the corrifol is corrifol 188, while the polysorbate is polysorbate 20 or polysorbate 80 surfactant. Most preferably, the surfactant is polysorbate 20.

[0178] The pharmaceutical composition preferably contains 0.01 to 2 mg / mL of a surfactant, 0.05 to 1.5 mg / mL of a surfactant, 0.1 to 0.6 mg / mL of a surfactant, 0.25 to 0.75 mg / mL of a surfactant, 0.4 to 0.6 mg / mL of a surfactant, 0.4 to 1.2 mg / mL of a surfactant, or 0.8 to 1.1 mg / mL of a surfactant.

[0179] The pharmaceutical composition preferably contains the surfactant at a molar ratio of surfactant to fusion protein of 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.

[0180] The pharmaceutical composition preferably contains 0.01 to 2 mg / mL polysorbate 20, 0.05 to 1.5 mg / mL polysorbate 20, 0.05 to 0.3 mg / mL polysorbate 20, 0.05 to 0.15 mg / mL polysorbate 20, 0.1 to 0.7 mg / mL polysorbate 20, 0.3 to 0.7 mg / mL polysorbate 20, 0.4 to 0.6 mg / mL polysorbate 20, 0.4 to 1.3 mg / mL polysorbate 20, 0.8 to 1.2 mg / mL polysorbate 20, 0.9 to 1.1 mg / mL polysorbate 20, or most preferably about 0.5 mg / mL polysorbate 20.

[0181] The pharmaceutical composition preferably contains polysorbate 20 at a molar ratio of polysorbate 20 to fusion protein of 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.

[0182] Unless only one surfactant is specified, the amount and concentration of that surfactant refers to the sum of the amounts and concentrations of all surfactants. The concentration of that surfactant or any particular surfactant can be substituted by a molar ratio.

[0183] sugar component The pharmaceutical composition preferably contains a sugar component, such as a cryoprotectant sugar component. In the context of the present invention, sugar components can serve one or more functions within the pharmaceutical composition. For example, they may serve as cryoprotectants during freeze-drying. The sugar component provides tonicity, e.g., osmolality, within a desired range (e.g., for isotonicity of an undiluted injectable formulation—e.g., an osmolality of 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, and most preferably 270-310 mOsmol / L). The use of a sugar component that contributes to tonicity may be particularly useful when high ionic strength is less desirable, although generally, compositions of the present invention tend to favor relatively high ionic strength due to the relatively high concentration of fusion protein. The sugar component may promote solubility of the fusion protein and / or other excipients within the pharmaceutical composition. The sugar moiety provides a stabilizing effect to the fusion protein of the pharmaceutical composition, e.g., improving conformational stability (e.g., reducing protein unfolding events that may 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 a sugar moiety (or any one or more of those specifically mentioned herein in connection with a sugar moiety).

[0184] Preferably, the sugar is non-ionic and / or does not contain ionizable groups.

[0185] In some embodiments, the sugar component is a non-reducing sugar component. The sugar component may also include one or more sugar(s) and / or sugar alcohol(s). In some embodiments, the sugar component includes one or more sugar(s) or one or more sugar alcohol(s). Preferably, the sugar component consists of a single compound. In other embodiments, the sugar component includes at most one sugar or at most one sugar alcohol.

[0186] Preferably, the sugar component is selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, more preferably from the group consisting of trehalose or sucrose.

[0187] In some embodiments, the sugar component is a sugar 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.

[0188] 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, arabitol, xylitol, ribitol, and inositol. In some embodiments, the sugar component is a sugar alcohol selected from mannitol or sorbitol.

[0189] The pharmaceutical composition preferably contains 30 to 400 mM sugar component, 40 to 300 mM sugar component, 40 to 100 mM sugar component, 40 to 60 mM sugar component, 90 to 290 mM sugar component, 100 to 200 mM sugar component, 130 to 170 mM sugar component, 200 to 300 mM sugar component, 230 to 270 mM sugar component, about 159 mM sugar component, about 100 mM sugar component, or about 50 mM sugar component.

[0190] The pharmaceutical composition preferably contains the sugar moiety at a molar ratio of sugar moiety to fusion protein of 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.

[0191] The pharmaceutical composition preferably contains 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 contains trehalose at a concentration of about 50 mM, about 75 mM, about 100 mM, or about 159 mM. The pharmaceutical composition preferably contains trehalose at a molar ratio of trehalose to fusion protein of 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, most preferably at a concentration of 40 to 200 mM.

[0192] The pharmaceutical composition preferably contains 50 to 300 mM sucrose, 150 to 290 mM sucrose, 220 to 280 mM sucrose, or 240 to 260 mM sucrose.

[0193] The pharmaceutical composition preferably contains 40 to 300 mM mannitol, 40 to 100 mM mannitol, 40 to 60 mM mannitol, 80 to 120 mM mannitol, 100 to 200 mM mannitol, 210 to 290 mM mannitol, or 240 to 260 mM mannitol.

[0194] The pharmaceutical composition preferably contains 40 to 300 mM sorbitol, 40 to 100 mM sorbitol, 40 to 60 mM sorbitol, 80 to 120 mM sorbitol, 100 to 200 mM sorbitol, 210 to 290 mM sorbitol, or 240 to 260 mM sorbitol.

[0195] Unless only one sugar component is specified, the amount and concentration for that sugar component refers to the sum of the amounts and concentrations of all sugar components. The sugar component or the concentration of any particular sugar component can be substituted by a molar ratio.

[0196] Amino acid components The pharmaceutical composition preferably contains an amino acid component. Amino acids may be used to adjust the osmolality of the pharmaceutical composition, e.g., to bring the osmolality within a preferred range (e.g., for isotonicity of an undiluted injectable formulation—e.g., an osmolality of 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, and most preferably 270-310 mOsmol / L). Amino acids can also promote the solubility of the fusion protein and / or other excipients in the pharmaceutical composition. Amino acids may also have a stabilizing effect on the fusion protein of the pharmaceutical composition, e.g., improving conformational stability (e.g., reducing protein unfolding events that may 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), may replace some or all of the tonicity agent (e.g., NaCl) and / or some or all of the sugar component (e.g., trehalose). Preferably, the pharmaceutical composition contains amino acid components in addition to histidine (wherein the histidine is used as or part of a buffering system) and / or methionine (wherein the methionine is used as or part of an antioxidant component).

[0197] However, the pharmaceutical composition may be characterized by the absence of an amino acid component (or any one or more specifically mentioned herein in connection with an amino acid component), preferably with the optional exception histidine (wherein histidine is used as or part of a buffering system), or with the optional exception methionine (wherein methionine is used as or part of an antioxidant component), or preferably with the optional exceptions histidine and methionine (wherein they are used as or part of a buffering system and as or part of an antioxidant component, respectively).

[0198] Preferably, the amino acid component comprises one or more amino acids, in particular one or more amino acids other than histidine (wherein histidine is, for example, included as or part of a buffer system) and / or one or more amino acids other than methionine (wherein methionine is, for example, included as or part of an antioxidant component). Preferably, the amino acid component comprises at most one amino acid, or at most one amino acid other than histidine (wherein histidine is, for example, included as or part of a buffer system) and / or methionine (wherein methionine is, for example, included as or part of an antioxidant component). Preferably, the amino acid component comprises a single amino acid, or a single amino acid other than histidine (wherein histidine is, for example, included as or part of a buffer system) and / or methionine (wherein methionine is, for example, included as or part of an antioxidant component). Preferably, the amino acid component comprises an L-amino acid. Preferably, the amino acid(s) making up this amino acid component are L-amino acid(s).

[0199] The amino acid component, or any amino acid therein, may be a pharmaceutically acceptable salt thereof, for example, in the case of arginine, the arginine may actually be provided in the form of arginine monohydrochloride.

[0200] Preferably, the amino acid component comprises an amino acid selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof. Preferably, the amino acid component comprises an amino acid selected from the group consisting of arginine, lysine, proline, glutamic acid, and any combination thereof.

[0201] Preferably, the amino acid component comprises arginine.

[0202] Preferably, the amino acid component comprises lysine.

[0203] Preferably, the amino acid component comprises proline.

[0204] Preferably, the amino acid component comprises glutamic acid.

[0205] The amino acid is preferably a charged amino acid (i.e., where the amino acid carries a net positive or negative charge). Preferably, the amino acid component comprises an amino acid salt (e.g., arginine hydrochloride).

[0206] The pharmaceutical composition preferably contains 10 to 300 mM of amino acid components, 20 to 260 mM of amino acid components, 30 to 110 mM of amino acid components, 30 to 60 mM of amino acid components, 35 to 95 mM of amino acid components, 40 to 170 mM of amino acid components, 50 to 200 mM of amino acid components, 60 to 140 mM of amino acid components, 110 to 190 mM of amino acid components, 140 to 180 mM of amino acid components, or approximately 50 mM of amino acid components.

[0207] The pharmaceutical composition preferably contains the amino acid component at a molar ratio of 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 of the amino acid component to the fusion protein.

[0208] The pharmaceutical composition preferably contains 20 to 300 mM arginine, 30 to 60 mM arginine, 30 to 50 mM arginine, 40 to 60 mM arginine, 60 to 80 mM arginine, 35 to 95 mM arginine, 80 to 120 mM arginine, 100 to 140 mM arginine, 120 to 180 mM arginine, 150 to 170 mM arginine, 200 to 300 mM arginine, or preferably about 50 mM arginine or about 75 mM arginine. The pharmaceutical composition preferably contains arginine at a molar ratio of arginine to fusion protein of 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.

[0209] The pharmaceutical composition preferably contains 20 to 200 mM lysine, 30 to 150 mM lysine, 30 to 80 mM lysine, 40 to 60 mM lysine, 60 to 90 mM lysine, 70 to 130 mM lysine, or 90 to 110 mM lysine.

[0210] The pharmaceutical composition preferably contains 20 to 200 mM proline, 30 to 150 mM proline, 30 to 80 mM proline, 40 to 60 mM proline, 60 to 90 mM proline, 70 to 130 mM proline, or 90 to 110 mM proline.

[0211] The pharmaceutical composition preferably contains 20 to 200 mM glutamic acid, 30 to 150 mM glutamic acid, 30 to 80 mM glutamic acid, 40 to 60 mM glutamic acid, 60 to 90 mM glutamic acid, 70 to 130 mM glutamic acid, or 90 to 110 mM glutamic acid.

[0212] Unless only one amino acid component is specified, the amount and concentration for that amino acid component refers to the sum of the amounts and concentrations of all amino acid components. The concentration of that amino acid component or any particular amino acid component can be substituted by a molar ratio.

[0213] Ionic strength The pharmaceutical compositions of the present invention preferably have a non-zero ionic strength. This ionic strength is believed to promote the solubility of the fusion protein, particularly the TGFβR portion thereof. A certain degree of ionic strength can reduce fragmentation. A certain degree of ionic strength can reduce aggregation.

[0214] Ionic strength may preferably be defined as the "molar ionic strength (I)" which is the ionic strength present in a given composition (which is preferably a solution, preferably an aqueous solution). All The molar ionic strength (I) is a function of the concentration of the ion. The molar ionic strength (I) may be defined by the following formula:

number

[0215] As an example, if NaCl is the only electrolyte contributing to the ionic strength of a composition, then the ionic strength is equal to the concentration of NaCl (i.e., 100 mM NaCl = 100 mM ionic strength) because each of the cations and anions in NaCl carries one charge - so for a 60 mM NaCl solution, the ionic strength is:

number

[0216] When a salt such as MgSO is the only electrolyte contributing to the ionic strength of a composition, its ionic strength is four times that of an equimolar concentration of sodium chloride because the cation and anion each have two charges - so for a 60 mM MgSO solution, the ionic strength is:

number

[0217] As a result, multiply charged ions contribute relatively more to the ionic strength than singly charged ions.

[0218] Amino acid salts (e.g., arginine monohydrochloride) can also contribute to ionic strength. Buffers (or their ion-buffering species—e.g., conjugate acids and / or conjugate bases) can also contribute to ionic strength.

[0219] The ionic strength of the composition is preferably equal to the sum of the ionic contributions (as defined by the formula above) from the tonicity agent and, if present, the buffer system. The ionic strength of the composition may be equal to any of the concentrations defined herein relative to the molarity of the tonicity agent plus any buffer system.

[0220] In some embodiments, the compositions of the present invention may be characterized by a non-buffering (or tonicity agent-based) ionic strength. In such cases, the non-buffering (or tonicity agent-based) ionic strength of the composition is preferably equal to any of the concentrations specified herein relative to the tonicity agent. Also in such cases, the ratio of the non-buffering (or tonicity agent-based) ionic strength to the molar concentration of the fusion protein is preferably the same as any of the molar ratios of tonicity agent to fusion protein as specified herein.

[0221] The pharmaceutical composition preferably has an ionic strength of 5 to 250 mM, 10 to 200 mM, 20 to 170 mM, 20 to 80 mM, 20 to 60 mM, 30 to 50 mM, 30 to 130 mM, 80 to 150 mM, 90 to 100 mM, 100 to 170 mM, 110 to 130 mM, 150 to 170 mM, 130 to 170 mM, about 40 mM, about 60 mM, or about 100 mM.

[0222] The pharmaceutical composition preferably has an ionic strength to fusion protein molar ratio of 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.

[0223] This ionic strength is preferably provided at least in part or entirely by sodium chloride. This ionic strength is preferably provided at least in part or entirely by sodium chloride and arginine (or a salt thereof). This ionic strength is preferably provided at least in part or entirely by sodium chloride and / or an amino acid salt (e.g., arginine or an arginine salt).

[0224] Isotonic agent The pharmaceutical composition preferably contains a tonicity agent, such as a non-buffering tonicity agent (i.e., an isotonicity agent that does not provide a pH buffering effect, unlike buffer salts that themselves contribute to overall tonicity to some extent). In the context of the present invention, the tonicity agent may be used to adjust the osmolality of the pharmaceutical composition, for example, to bring the osmolality within a preferred range (e.g., isotonicity of an undiluted injectable formulation—e.g., an osmolality of 200-400 mOsmol / L, more preferably 250-350 mOsmol / L, most preferably 270-310 mOsmol / L). Similarly, tonicity agents, particularly ionic tonicity agents (e.g., salt tonicity agents), may be used to provide ionic strength. The tonicity agent may also promote the solubility of the fusion protein and / or other excipients in the pharmaceutical composition. The tonicity agent also provides a stabilizing effect to the fusion protein of the pharmaceutical composition, e.g., improving conformational stability (e.g., reducing protein unfolding events that may increase the likelihood of eventual aggregation), reducing aggregation, and / or reducing fragmentation.

[0225] However, in some embodiments, the pharmaceutical composition is characterized by the absence of a (non-buffering) tonicity agent (or any one or more specifically mentioned herein in connection with a tonicity agent). Alternatively, some tonicity agents, particularly non-buffering salt tonicity agents (e.g., NaCl), may be replaced by some amino acid components and / or some sugar components, especially if said components themselves provide a tonicity effect.

[0226] The tonicity agent may be or include an isotonic cryoprotectant. An isotonic cryoprotectant is a compound or compounds that provide tonicity to a composition while also acting as a cryoprotectant, for example, during freeze-drying (particularly during its dry phase). Cryoprotectant activity is well known in the art and may include, for example, a compound(s) that stabilizes active ingredients and / or stabilizes the lyophilized cake during the dry phase of freeze-drying (e.g., prevents its collapse during drying). The isotonic cryoprotectant may comprise or consist of a sugar or sugar polyol, such as a sugar or sugar polyol defined herein in connection with the "sugar component." The isotonic cryoprotectant may comprise or consist of an amino acid, such as an "amino acid component" defined herein. Trehalose is an example of an isotonic cryoprotectant. Arginine can also act as a cryoprotectant. It may be advantageous for a composition to include a combination of both trehalose and arginine.

[0227] Preferably, the tonicity agent comprises an isotonicity agent selected from the group consisting of a salt isotonicity agent, a metal salt isotonicity agent, a non-buffered salt isotonicity agent, a metal halide salt isotonicity agent, an alkali metal or alkaline earth metal halide salt isotonicity agent, an alkali metal halide salt isotonicity agent, and an alkali metal halide salt isotonicity agent selected from sodium chloride or potassium chloride, preferably sodium chloride, and any combination thereof.

[0228] Preferably, the tonicity agent is an ionic tonicity agent and therefore contributes directly to the ionic strength of the composition. Preferably, the tonicity agent is a non-buffering ionic tonicity agent.

[0229] The pharmaceutical composition preferably contains 5 to 250 mM of an isotonic agent, 10 to 200 mM of an isotonic agent, 20 to 170 mM of an isotonic agent, 20 to 80 mM of an isotonic agent, 20 to 60 mM of an isotonic agent, 50 to 70 mM of an isotonic agent, 30 to 50 mM of an isotonic agent, 30 to 130 mM of an isotonic agent, 80 to 150 mM of an isotonic agent. The tonicity adjusting agent contains 90 to 110 mM of an isotonic agent, 90 to 100 mM of an isotonic agent, 100 to 170 mM of an isotonic agent, 110 to 130 mM of an isotonic agent, 150 to 170 mM of an isotonic agent, 130 to 170 mM of an isotonic agent, about 40 mM of an isotonic agent, about 60 mM of an isotonic agent, or about 100 mM of an isotonic agent.

[0230] The pharmaceutical composition preferably contains the isotonic agent at a molar ratio of isotonic agent to fusion protein of 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.

[0231] The pharmaceutical composition preferably contains 5 to 250 mM sodium chloride, 10 to 200 mM sodium chloride, 20 to 170 mM sodium chloride, 30 to 90 mM sodium chloride, 20 to 60 mM sodium chloride, 30 to 50 mM sodium chloride, 50 to 70 mM sodium chloride, 35 to 45 mM sodium chloride, 30 to 130 mM sodium chloride, 80 to 150 mM sodium chloride, 90 to 110 mM sodium chloride, 100 to 200 mM sodium chloride, 110 to 130 mM sodium chloride, 150 to 170 mM sodium chloride, 130 to 170 mM sodium chloride, about 40 mM sodium chloride, about 60 mM sodium chloride, or about 100 mM sodium chloride.

[0232] The pharmaceutical composition preferably contains sodium chloride at a molar ratio of sodium chloride to fusion protein of 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.

[0233] Unless only one tonicity agent is specified, the amount and concentration of this tonicity agent refers to the sum of the amounts and concentrations of all tonicity agents. The tonicity agent or concentrations of any particular tonicity agent can be substituted by molar ratios.

[0234] Generally, in the context of the formulations of the present invention, the only viable component for providing ionic strength, excluding the buffer system, is a tonicity agent(s), such as sodium chloride and / or a charged amino acid (e.g., arginine, or arginine hydrochloride, or other amino acid salts). NaCl as a tonicity agent is considered a particularly advantageous component, since it generally promotes solubility of the fusion protein in aqueous media, but it may also participate in charge shielding, particularly of certain groups on the receptor portion of the fusion protein. Experiments often reveal that as the concentration of the fusion protein increases, the formulation generally benefits from higher concentrations of NaCl. For example, 5-15 mg / mL fusion protein may benefit from 30-50 mM NaCl; 35-55 mg / mL fusion protein may benefit from 50-70 mM NaCl; and 50-70 mg / mL fusion protein may benefit from 130-170 mM NaCl.

[0235] antioxidants The pharmaceutical composition preferably contains an antioxidant. The antioxidant can inhibit oxidative degradation pathways open to the fusion protein and / or other components in the pharmaceutical composition. For example, the antioxidant may inhibit oxidation of the fusion protein, e.g., by inhibiting oxidation of certain oxidizable amino acid residues in the fusion protein, and / or inhibit oxidative deamination pathways.

[0236] However, in some embodiments, the pharmaceutical composition is characterized by the absence of an antioxidant (or any one or more specifically mentioned herein in connection with an antioxidant).

[0237] Preferably, the antioxidant comprises an antioxidant selected from the group consisting of amino acid or peptide antioxidants, metal antioxidants, vitamin antioxidants, carotenoid antioxidants, polyphenol antioxidants, aromatic or phenolic antioxidants, chelating antioxidants, thiol antioxidants, and any combination thereof.

[0238] Preferably, the antioxidant comprises a single compound antioxidant.

[0239] Preferably, the antioxidant comprises an amino acid antioxidant, such as an amino acid antioxidant selected from the group consisting of methionine, N-acetyl-l-cysteine, cysteine, and glutathione.

[0240] Most preferably, the antioxidant is methionine.

[0241] The pharmaceutical composition preferably contains 0.1 to 50 mM of an antioxidant, 1 to 30 mM of an antioxidant, 2 to 20 mM of an antioxidant, 3 to 10 mM of an antioxidant, or 4 to 6 mM of an antioxidant.

[0242] The pharmaceutical composition preferably contains antioxidant to fusion protein in a molar ratio of 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.

[0243] The pharmaceutical composition preferably contains 1 to 30 mM methionine, 2 to 20 mM methionine, 3 to 12 mM methionine, 4 to 6 mM methionine, or about 5 mM methionine.

[0244] The pharmaceutical composition preferably contains methionine at a molar ratio of methionine to fusion protein of 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.

[0245] Unless only one antioxidant is specified, the amount and concentration of this antioxidant refers to the sum of the amounts and concentrations of all antioxidants. The antioxidant or concentrations of any particular antioxidant can be expressed in terms of molar ratios.

[0246] chelating agents The pharmaceutical composition may also contain a chelating agent. In the context of the present invention, a chelating agent such as EDTA (other chelating agents are well known in the art and may serve similar or identical functions) may increase the stability of the pharmaceutical composition and, in particular, inhibit degradation of the fusion protein. For example, a chelating agent may reduce the likelihood of aggregation and may also act as an antioxidant. A chelating agent may also sequester residual metals that would otherwise promote degradation of the fusion protein and / or other components present in the pharmaceutical composition.

[0247] In some embodiments, however, the pharmaceutical composition is characterized by the absence of a chelating agent (or any one or more specifically mentioned herein in connection with a chelating agent).

[0248] Preferably, the chelating agent comprises EDTA or EGTA, and more preferably, the chelating agent comprises EDTA.

[0249] The pharmaceutical composition preferably contains 0.001 to 0.5 mM of the chelating agent, 0.01 to 0.2 mM of the chelating agent, or 0.025 to 0.075 mM of the chelating agent, and preferably contains the chelating agent to the fusion protein in a molar ratio of 10:1 to 1:550, 4:1 to 1:55, or 1.5:1 to 1:22.

[0250] The pharmaceutical composition preferably contains 0.001 to 0.5 mM EDTA, 0.01 to 0.2 mM EDTA, or 0.025 to 0.075 mM EDTA, and preferably contains EDTA at a molar ratio of EDTA to fusion protein of 10:1 to 1:550, 4:1 to 1:55, or 1.5:1 to 1:22.

[0251] Unless only one chelating agent is specified, the amount and concentration for that chelating agent refers to the sum of the amounts and concentrations of all chelating agents. The concentration of that chelating agent or any particular chelating agent can be substituted by a molar ratio.

[0252] Osmolality Generally, the osmolality of the pharmaceutical composition is not critical and is determined by the amount of osmolyte required to stabilize the formulation. When the pharmaceutical formulation is intended for injection, e.g., subcutaneous injection, without prior dilution, it is preferable that the formulation be substantially isotonic to avoid skin irritation. In some embodiments, the concentration of the fusion protein can be carefully balanced with the concentrations of the isotonic agent (e.g., NaCl), amino acid components (e.g., arginine / arginine hydrochloride), and sugar components (e.g., trehalose) to provide a composition with a desired osmolality. 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.

[0253] Specific Embodiments The examples and data presented herein take into account the various specific embodiments discussed below and suggest that certain technical effects may result from particular properties or combinations of properties.

[0254] pH Experimental results show that liquid compositions having a pH within 2 pH units, preferably within 1.5 pH units, and even more preferably within 1 pH unit of the pI of the TGFβR2 portion of the fusion protein are surprisingly stable. Preferably, the pH is 4 or greater, and more preferably 5 or greater. However, higher pH can lead to protein stability and solubility issues; therefore, the liquid compositions preferably have a pH of 7 or less, more preferably 6.3 or less. A particularly preferred pH range is pH 5.3-6.3.

[0255] The following numbered paragraphs E1-E12 reveal specific embodiments of the invention: E1. A pharmaceutical composition containing an IgG:TGFβR fusion protein and characterized by a pH that is within 2 pH units of the pI of the TGFβR portion of the fusion protein. E2. A pharmaceutical composition containing an IgG:TGFβR fusion protein and characterized by a pH that is within 1.5 pH units of the pI of the TGFβR portion of the fusion protein. E3. A pharmaceutical composition containing an IgG:TGFβR fusion protein and characterized by a pH that is within 1 pH unit of the pI of the TGFβR portion of the fusion protein. E4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by a pH of 4-7. E5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by a pH of 5.2 to 6.1. E6. The pharmaceutical composition of any of E1-E5, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. E7. Any of the pharmaceutical compositions E1 to E6, further comprising an isotonic agent, preferably sodium chloride. E8. The pharmaceutical composition of any one of E1 to E7, further comprising a surfactant, preferably polysorbate 20. E9. The pharmaceutical composition of any one of E1 to E8, further comprising a sugar component, preferably trehalose. E10. Any of the pharmaceutical compositions E1 to E9, further comprising an amino acid component, preferably arginine and / or lysine. E11. The pharmaceutical composition of any of E1 to E10, further comprising a buffer system, preferably a histidine buffer system. E12. Any of the pharmaceutical compositions E1 to E11, further comprising an antioxidant, preferably methionine.

[0256] Aqueous formulation and ionic strength Formulation studies revealed particular aqueous solubility challenges for the IgG:TGFβR fusion protein, which, without wishing to be bound by theory, are believed to stem from the significant difference between the pI of the antibody portion and the pI of the fused TGFβR portion. Surprisingly, ionic strength can have a dramatic effect on the aqueous solubility of this fusion protein. Ionic strength also appears to play a role in protein stabilization.

[0257] During the early phases of formulation research, lyophilized formulations were considered the only viable formulation option from the standpoint of long-term storage stability, and as discussed below, these appeared to impose certain limitations. However, as research progressed, liquid formulations were found to be also viable and generally preferable for practical reasons (e.g., they avoid energy-intensive freeze-drying and manual reconstitution by the physician).

[0258] As mentioned above under "ionic strength," the present compositions may be characterized by an ionic strength that preferably includes contributions from all ionic species within the composition. However, in some embodiments, the present compositions may be characterized by a non-buffering (or tonicity agent-based) ionic strength, in which case the ionic strength preferably includes contributions from all ionic species except those of the buffer system. Because buffer systems are used at relatively low concentrations, the following embodiments may apply to either definition of ionic strength.

[0259] Preferably, the ionic strength of the composition is at least 20 mM, whereas in the case of pharmaceutical compositions intended for subcutaneous use without pre-dilution, the maximum value is preferably dictated by the requirement of osmolality, in order to avoid any excessive hypertonicity. In a preferred embodiment, the maximum ionic strength is 200 mM, more preferably 160 mM.

[0260] Ionic strength can be provided by a variety of charged or ionic excipients, but suitably ionic strength is provided by one or more ionic tonicity agents, such as sodium chloride, and / or charged amino acids (e.g., arginine or arginine salts), with sodium chloride being most preferred.

[0261] Sodium chloride may be the sole non-buffering contributor to ionic strength. In some embodiments, the pharmaceutical composition contains an amino acid component, which is defined separately from the tonicity agent (e.g., NaCl), and which may include a charged or ionic compound, such as arginine or an arginine salt. In such cases, it will be understood that the amino acid component contributes to the overall ionic strength and, indeed, tonicity.

[0262] The following numbered paragraphs F1-F12 identify specific embodiments of the present invention: F1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by an ionic strength of 20 mM or greater (optionally a non-buffering ionic strength). F2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by an ionic strength of 20 mM or greater (optionally non-buffering ionic strength) and an osmolality of 255-345 mOsm / kg. F3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by an ionic strength between 20 and 200 mM (optionally non-buffering ionic strength). F4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent. F5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more sodium chloride. F6. The pharmaceutical composition of any one of F1 to F5, further characterized by a pH defined in any one of E1 to E5. F7. The pharmaceutical composition of any of F1-F6, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. F8. The pharmaceutical composition of any one of F1 to F7, further comprising a surfactant, preferably polysorbate 20. F9. The pharmaceutical composition of any one of F1 to F8, further comprising a sugar component, preferably trehalose. F10. The pharmaceutical composition of any one of F1 to F9, further comprising an amino acid component, preferably arginine and / or lysine. F11. The pharmaceutical composition of any of F1 to F10, further comprising a buffer system, preferably a histidine buffer system. F12. The pharmaceutical composition of any one of F1 to F11, further comprising an antioxidant, preferably methionine.

[0263] Lyophilization and Ionic Strength Initial research focused on lyophilized formulations, which at the time appeared to be the most viable option 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. As discussed in the previous section, "Aqueous Formulations and Ionic Strength," ionic strength, particularly when provided in the composition in the form of sodium chloride, became a tool for meeting the requirements of tonicity, stability, and solubility. However, research has shown that the use of too much sodium chloride can impair freeze-drying and cause collapse of the lyophilized cake. While the inclusion of a cryoprotectant such as trehalose mitigates this to some extent, there appears to be a maximum tolerable amount of sodium chloride that meets each of the above goals of a lyophilized formulation. As discussed below, higher protein concentrations generally require the support of higher concentrations of sodium chloride, so this limitation on the amount of sodium chloride also potentially limited the concentration of this fusion protein.

[0264] The following numbered paragraphs G1-G21 identify specific embodiments of the present invention: G1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by an ionic strength of 20-50 mM (optionally a non-buffering ionic strength). G2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and characterized by an ionic strength of 20-50 mM (optionally non-buffering ionic strength) and an osmolality of 255-345 mOsm / kg. G3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 to 50 mM of an ionic tonicity agent. G4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 to 50 mM sodium chloride. G5. Any of the pharmaceutical compositions G1 to G4, wherein the concentration of the ionic tonicity agent (or sodium chloride) is 35 to 45 mM. G6. Any of the pharmaceutical compositions G1 to G4, wherein the concentration of the ionic tonicity agent (or sodium chloride) is about 40 mM. G7. The pharmaceutical composition of any one of G1 to G6, wherein the concentration of the IgG:TGFβR fusion protein is 5 to 25 mM. G8. Any of the pharmaceutical compositions G1 to G7, wherein the concentration of the IgG:TGFβR fusion protein is about 10 mM. G9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ionic tonicity agent, at a molar ratio of ionic tonicity agent to fusion protein of 180:1 to 9110:1. G10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an ionic tonicity agent, at a molar ratio of the ionic tonicity agent to the fusion protein of 360:1 to 911:1. G11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and sodium chloride, at a molar ratio of sodium chloride to fusion protein of 180:1 to 9110:1. G12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and sodium chloride at a molar ratio of sodium chloride to fusion protein of 360:1 to 911:1. G13. A pharmaceutical composition according to any one of G1 to G12, further characterized by a pH defined in any one of E1 to E5. G14. The pharmaceutical composition of any of G1 to G13, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. G15. Any of the pharmaceutical compositions G1 to G14, further containing a surfactant, preferably polysorbate 20. G16. Any of the pharmaceutical compositions G1 to G15, further comprising a sugar component, preferably trehalose. G17. Any of the pharmaceutical compositions G1 to G16, further comprising an amino acid component, preferably arginine and / or lysine. G18. The pharmaceutical composition of any of G1 to G17, further comprising a buffer system, preferably a histidine buffer system. G19. Any of the pharmaceutical compositions G1 to G18, further containing an antioxidant, preferably methionine. G20. Any of the pharmaceutical compositions G1 to G19, which is a reconstituted lyophilized pharmaceutical composition. G21. Any of the pharmaceutical compositions of G1 to G19, which is a solid, lyophilized pharmaceutical composition suitable for reconstitution.

[0265] Isotonic cryoprotectant For lyophilized formulations, the inclusion of an isotonic cryoprotectant has been found to help achieve the right balance between solubility and isotonicity (in liquid form), stability (in lyophilized and reconstituted liquid form), and lyophilizability. Unlike ionic strength providers such as sodium chloride, which damage the lyophilized cake, isotonic cryoprotectants can provide tonicity without impairing freeze-drying ability, thereby allowing the use of minimal but sufficiently high amounts of ionic strength providers (e.g., NaCl) to promote protein solubility and stability. In many cases, the use of an isotonic cryoprotectant can at least partially mitigate the negative effects of ionic tonicity agents (e.g., NaCl) during freeze-drying.

[0266] Suitable isotonic cryoprotectants include sugar moieties (multiple) and / or amino acid moieties (multiple), such as trehalose and / or arginine, with sugar moieties (preferably a single sugar moiety) being more preferred. Preferred sugar moieties are described herein, with the most preferred sugar moiety being trehalose.

[0267] The following numbered paragraphs H1-H34 identify specific embodiments of the present invention: H1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (eg, a sugar moiety and / or an amino acid moiety as defined above), and an ionic tonicity agent. H2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component as defined above), and an ionic tonicity agent, in a molar ratio of isotonic cryoprotectant to ionic tonicity agent of 10:1 to 1:2. H3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and sodium chloride in a molar ratio of trehalose to sodium chloride of 10:1 to 1:2. H4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (eg, a sugar moiety and / or an amino acid moiety as defined above), and 20-50 mM of an ionic tonicity agent. H5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component), and 20-50 mM sodium chloride. H6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and 20-50 mM of an ionic tonicity agent. H7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and 20-50 mM sodium chloride. H8. The pharmaceutical composition of any of H1 to H7, wherein the isotonic cryoprotectant is present at a concentration of 100 to 200 mM, preferably 130 to 170 mM. H9. The pharmaceutical composition of any one of H1 to H8, wherein the concentration of the IgG:TGFβR fusion protein is 5 to 25 mM. H10. The pharmaceutical composition of any of H1 to H9, wherein the concentration of the IgG:TGFβR fusion protein is about 10 mM. H11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component), and an ionic tonicity agent, in a molar ratio of ionic tonicity agent to fusion protein of 180:1 to 9110:1. H12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component), and an ionic tonicity agent, in a molar ratio of ionic tonicity agent to fusion protein of 360:1 to 911:1. H13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component), and sodium chloride, in a molar ratio of sodium chloride to fusion protein of 180:1 to 9110:1. H14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an isotonic cryoprotectant (e.g., a sugar component and / or an amino acid component), and sodium chloride, in a molar ratio of sodium chloride to fusion protein of 360:1 to 911:1. H15. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and an ionic tonicity agent, at a molar ratio of ionic tonicity agent to fusion protein of 180:1 to 9110:1. H16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and an ionic tonicity agent, at a molar ratio of ionic tonicity agent to fusion protein of 360:1 to 911:1. H17. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and sodium chloride, at a molar ratio of sodium chloride to fusion protein of 180:1 to 9110:1. H18. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and sodium chloride, at a molar ratio of sodium chloride to fusion protein of 360:1 to 911:1. H19. The pharmaceutical composition of any of H1 to H18, wherein the isotonic cryoprotectant is selected from the group consisting of trehalose, sucrose, sorbitol, mannitol, arginine, and any combination thereof. H20. Any of the pharmaceutical compositions H1 to H19, wherein the isotonic cryoprotectant is a combination of trehalose and arginine. H21. Any of the pharmaceutical compositions H1 to H20, wherein the isotonic cryoprotectant is trehalose alone. H22. The pharmaceutical composition of any one of H1 to H21, wherein the molar ratio of the isotonic cryoprotectant to the ionic tonicity agent to the fusion protein is 910-36430:180-9110:1. H23. The pharmaceutical composition of any one of H1 to H22, wherein the molar ratio of isotonic cryoprotectant to ionic tonicity agent to fusion protein is 1820-3643:360-911:1. H24. The pharmaceutical composition of any one of H1 to H23, wherein the molar ratio of trehalose to sodium chloride to the fusion protein is 1820 to 3643:360 to 911:1. H25. A pharmaceutical composition according to any one of H1 to H24, further characterized by a pH as defined in any one of E1 to E5. H26. The pharmaceutical composition of any of H1 to H25, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. H27. Any of the pharmaceutical compositions of H1 to H26, further comprising a surfactant, preferably polysorbate 20. H28. The pharmaceutical composition of any one of H1 to H27, further comprising a sugar component, preferably trehalose. H29. The pharmaceutical composition of any one of H1 to H28, further comprising an amino acid component, preferably arginine and / or lysine. H30. The pharmaceutical composition of any of H1 to H29, further comprising a buffer system, preferably a histidine buffer system. H31. Any of the pharmaceutical compositions of H1 to H30, further comprising an antioxidant, preferably methionine. H32. Any of the pharmaceutical compositions of H1 to H31, which is a reconstituted lyophilized pharmaceutical composition. H33. The pharmaceutical composition of any of H1 to H31, which is a solid lyophilized pharmaceutical composition suitable for reconstitution. H34. The pharmaceutical composition of any of H1-H33, wherein the ionic tonicity agent is a non-buffering ionic tonicity agent.

[0268] pH and ionic strength As mentioned above, ionic strength (especially when provided by NaCl) can improve the solubility of fusion proteins at all pHs, but this effect is particularly pronounced at relatively high pHs.

[0269] The following numbered paragraphs I1-I14 identify specific embodiments of the present invention: I1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent, and characterized by a pH of 5-7. I2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more sodium chloride, and characterized by a pH of 5-7. I3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent, and characterized by a pH of 5.4 to 6.4. I4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more sodium chloride, and characterized by a pH of 5.4 to 6.4. I5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent, and characterized by a pH of 5.8 to 6.8. I6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 20 mM or more sodium chloride, and characterized by a pH of 5.8 to 6.8. I7. The pharmaceutical composition of any of I1 to I6, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. I8. Any of the pharmaceutical compositions I1 to I7, further comprising a surfactant, preferably polysorbate 20. I9. The pharmaceutical composition of any one of I1 to I8, further comprising a sugar component, preferably trehalose. I10. A pharmaceutical composition according to any one of I1 to I9, further comprising an amino acid component, preferably arginine and / or lysine. I11. The pharmaceutical composition of any of I1 to I10, further comprising a buffer system, preferably a histidine buffer system. I12. Any of the pharmaceutical compositions I1 to I11, further comprising an antioxidant, preferably methionine. I13. Any of the pharmaceutical compositions I1 to I12, which is a liquid pharmaceutical composition. I14. The pharmaceutical composition of any of I1 to I13, wherein the ionic tonicity agent is a non-buffering ionic tonicity agent.

[0270] Fusion protein concentration Although early studies suggested that this fusion protein concentration may be limited in lyophilized formulations due to the maximum amount of sodium chloride that can withstand the freeze-drying process, subsequent studies revealed the long-term viability of liquid formulations, which in turn raised the possibility for higher fusion protein concentrations (especially when appropriately supported by increased ionic strength).

[0271] The following numbered paragraphs J1-J12 reveal specific embodiments of the present invention: J1. A pharmaceutical composition containing greater than 20 mg / mL, preferably greater than 80 mg / mL, of an IgG:TGFβR fusion protein. J2. A pharmaceutical composition comprising greater than 20 mg / mL, preferably greater than 80 mg / mL, of an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent. J3. A pharmaceutical composition comprising greater than 20 mg / mL, preferably greater than 80 mg / mL, of an IgG:TGFβR fusion protein and 20 mM or more of an ionic tonicity agent, and characterized by a pH of 4-8. J4. The pharmaceutical composition of any of J1 to J3, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. J5. A pharmaceutical composition according to any one of J1 to J4, characterized by a pH defined in any one of E1 to E5. J6. Any of the pharmaceutical compositions J1 to J5, further comprising an isotonic agent, preferably sodium chloride. J7. The pharmaceutical composition of any one of J1 to J6, further comprising a surfactant, preferably polysorbate 20. J8. The pharmaceutical composition of any one of J1 to J7, further comprising a sugar component, preferably trehalose. J9. A pharmaceutical composition according to any one of J1 to J8, further comprising an amino acid component, preferably arginine and / or lysine. J10. The pharmaceutical composition of any one of J1 to J9, further comprising a buffer system, preferably a histidine buffer system. J11. Any of the pharmaceutical compositions J1 to J10, further comprising an antioxidant, preferably methionine. J12. Any of the pharmaceutical compositions of J1 to J11, which is a liquid pharmaceutical composition.

[0272] Fusion protein concentration and ionic strength Studies have shown that relatively high concentrations of fusion protein can be achieved, preferably aided by increased ionic strength. Increased ionic strength (particularly sodium chloride concentration) in conjunction with fusion protein concentration ensures solubility and stability of larger proteins. In some cases, sugar components such as trehalose can partially compensate for the lack of NaCl, and the presence of ionic strength providers has generally been found to be preferable.

[0273] The following numbered paragraphs K1 to K27 reveal specific embodiments of the present invention: K1. A pharmaceutical composition comprising 5-150 mg / mL of an IgG:TGFβR fusion protein and further characterized by an ionic strength of 5-250 mM (optionally a non-buffering ionic strength). K2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and further characterized by an ionic strength to fusion protein molar ratio of 1000:1 to 70:1. K3. A pharmaceutical composition comprising 5-25 mg / mL of an IgG:TGFβR fusion protein and further characterized by an ionic strength of 20-50 mM (optionally a non-buffering ionic strength). K4. A pharmaceutical composition comprising 25-45 mg / mL of an IgG:TGFβR fusion protein and further characterized by an ionic strength of 40-80 mM (optionally a non-buffering ionic strength). K5. A pharmaceutical composition containing at least 25 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of at least 50 mM (optionally non-buffering ionic strength). K6. A pharmaceutical composition comprising 45-65 mg / mL of an IgG:TGFβR fusion protein and further characterized by an ionic strength of 80-170 mM (optionally non-buffering ionic strength). K7. A pharmaceutical composition containing 45-65 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 80-120 mM (optionally non-buffering ionic strength), preferably wherein a sugar component (most preferably trehalose) is also present. K8. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and further characterized by an ionic strength of 120-170 mM (optionally non-buffering ionic strength), preferably in the absence of a sugar moiety (most preferably trehalose). K9. A pharmaceutical composition comprising 65-115 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 100-200 mM (optionally non-buffering ionic strength). K10. A pharmaceutical composition comprising 5 to 150 mg / mL of an IgG:TGFβR fusion protein and 10 to 200 mM of an isotonic agent (preferably 10 to 200 mM of sodium chloride). K11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an isotonicity agent (preferably sodium chloride) at a molar ratio of the isotonicity agent (preferably sodium chloride) to the fusion protein of 910:1 to 200:1. K12. A pharmaceutical composition comprising 5 to 25 mg / mL of an IgG:TGFβR fusion protein and 20 to 50 mM of an isotonic agent (preferably 20 to 50 mM of sodium chloride). K13. A pharmaceutical composition comprising 25 to 45 mg / mL of an IgG:TGFβR fusion protein and 40 to 80 mM of an isotonic agent (preferably 40 to 80 mM of sodium chloride). K14. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein and at least 50 mM of a tonicity agent (preferably at least 50 mM sodium chloride). K15. A pharmaceutical composition comprising 45 to 65 mg / mL of an IgG:TGFβR fusion protein and 80 to 170 mM of an isotonic agent (preferably 80 to 170 mM of sodium chloride). K16. A pharmaceutical composition comprising 45-65 mg / mL IgG:TGFβR fusion protein and 80-120 mM of an isotonicity agent (preferably 80-120 mM sodium chloride), preferably in which a sugar component (most preferably trehalose) is also present. K17. A pharmaceutical composition comprising 45-65 mg / mL of an IgG:TGFβR fusion protein and 120-170 mM of an isotonic agent (preferably 120-170 mM sodium chloride), preferably in the absence of a sugar component (most preferably trehalose). K18. A pharmaceutical composition comprising 65 to 115 mg / mL of an IgG:TGFβR fusion protein and 100 to 200 mM of an isotonic agent (preferably 100 to 200 mM sodium chloride). K19. The pharmaceutical composition of any of K1 to K18, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. K20. A pharmaceutical composition according to any one of K1 to K19, characterized by a pH defined in any one of E1 to E5. K21. Any of the pharmaceutical compositions K1 to K20, further comprising an isotonic agent, preferably sodium chloride. K22. The pharmaceutical composition of any one of K1 to K21, further comprising a surfactant, preferably polysorbate 20. K23. A pharmaceutical composition according to any one of K1 to K22, further comprising a sugar component, preferably trehalose. K24. A pharmaceutical composition according to any one of K1 to K23, further comprising an amino acid component, preferably arginine and / or lysine. K25. The pharmaceutical composition of any of K1 to K24, further comprising a buffer system, preferably a histidine buffer system. K26. Any of the pharmaceutical compositions K1 to K25, further comprising an antioxidant, preferably methionine. K27. Any of the pharmaceutical compositions K1 to K26, which is a liquid pharmaceutical composition.

[0274] Fusion protein concentration and pH In general, it has been found that relatively high concentrations of fusion protein tend to be best supported by a slightly higher pH, albeit at a pH even closer to the pI of the TGFβR moiety than would normally be expected.

[0275] The following numbered paragraphs L1 to L17 reveal specific embodiments of the present invention: L1. A pharmaceutical composition comprising 5 to 150 mg / mL of an IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 7.5, preferably a pH of 5.0 to 7.0. L2. A pharmaceutical composition comprising 5 to 150 mg / mL of an IgG:TGFβR fusion protein and characterized by a pH of 5.3 to 6.3, and further characterized by the absence of acetate buffer and acetate salts. L3. A pharmaceutical composition comprising 5 to 150 mg / mL of an IgG:TGFβR fusion protein, a histidine buffer system, and characterized by a pH of 5.3 to 6.3. L4. A pharmaceutical composition comprising 5 to 25 mg / mL of an IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 6.5, preferably a pH of 5.0 to 5.7. L5. A pharmaceutical composition containing 25 to 45 mg / mL of IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 7.5, preferably a pH of 5.3 to 6.2. L6. A pharmaceutical composition containing at least 25 mg / mL of IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 7.5, preferably a pH of 5.3 to 6.5. L7. A pharmaceutical composition containing at least 35 mg / mL of IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 7.5, preferably a pH of 5.4 to 7.0. L8. A pharmaceutical composition comprising 45 to 65 mg / mL of an IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 7.5, preferably a pH of 5.5 to 6.5. L9. A pharmaceutical composition comprising 65 to 115 mg / mL of an IgG:TGFβR fusion protein and characterized by a pH of 5.0 to 8.0, preferably a pH of 5.8 to 6.8. L10. The pharmaceutical composition of any of L1-L9, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. L11. Any of the pharmaceutical compositions of L1 to L10, further comprising an isotonic agent, preferably sodium chloride. L12. Any of the pharmaceutical compositions of L1 to L11, further comprising a surfactant, preferably polysorbate 20. L13. The pharmaceutical composition of any one of L1 to L12, further comprising a sugar component, preferably trehalose. L14. The pharmaceutical composition of any one of L1 to L13, further comprising an amino acid component, preferably arginine and / or lysine. L15. The pharmaceutical composition of any of L1 to L14, further comprising a buffer system, preferably a histidine buffer system. L16. Any of the pharmaceutical compositions L1 to L15, further comprising an antioxidant, preferably methionine. L17. Any of the pharmaceutical compositions of L1 to L16, which is a liquid pharmaceutical composition.

[0276] Fusion protein concentration, ionic strength, and pH As alluded to above, relatively high concentrations of fusion protein tend to be best supported by slightly higher pH and slightly higher ionic strength.

[0277] The following numbered paragraphs M1-M28 reveal specific embodiments of the present invention: M1. A pharmaceutical composition containing 5-150 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 5-250 mM (optionally non-buffering ionic strength) and a pH of 5.0-7.0. M2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and further characterized by an ionic strength to fusion protein molar ratio of 1000:1 to 70:1, and a pH of 5.0 to 6.5. M3. A pharmaceutical composition containing 5 to 150 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength to fusion protein molar ratio of 375:1 to 150:1, and a pH of 5.3 to 6.3. M4. A pharmaceutical composition containing 5-25 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 20-50 mM (optionally non-buffering ionic strength) and a pH of 5.0-6.5, preferably pH 5.0-5.7. M5. A pharmaceutical composition containing 25-45 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 40-80 mM (optionally non-buffering ionic strength) and a pH of 5.0-7.5, preferably pH 5.3-6.2. M6. A pharmaceutical composition containing at least 25 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of at least 50 mM (optionally non-buffering ionic strength), and a pH of 5.0 to 7.5, preferably 5.3 to 6.5. M7. A pharmaceutical composition containing 45-65 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 80-170 mM (optionally non-buffering ionic strength) and a pH of 5.0-7.5, preferably pH 5.5-6.5. M8. A pharmaceutical composition containing 45-65 mg / mL of IgG:TGFβR fusion protein, and further characterized by an ionic strength of 80-120 mM (optionally non-buffering ionic strength), and a pH of 5.0-7.5, preferably pH 5.5-6.5, and preferably wherein a sugar component (most preferably trehalose) is also present. M9. A pharmaceutical composition comprising 45-65 mg / mL of IgG:TGFβR fusion protein, further characterized by an ionic strength of 120-170 mM (optionally non-buffering ionic strength), and a pH of 5.0-7.5, preferably pH 5.5-6.5, preferably in the absence of a sugar component (most preferably trehalose). M10. A pharmaceutical composition containing 65-115 mg / mL of IgG:TGFβR fusion protein and further characterized by an ionic strength of 100-200 mM (optionally non-buffering ionic strength), and a pH of 5.0-8.0, preferably pH 5.8-6.8. M11. A pharmaceutical composition comprising 5 to 150 mg / mL of an IgG:TGFβR fusion protein and 10 to 200 mM of an isotonic agent (preferably 10 to 200 mM sodium chloride), and further characterized by a pH of 5 to 7. M12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a tonicity agent (preferably sodium chloride) in a molar ratio of tonicity agent (preferably sodium chloride) to fusion protein of 910:1 to 200:1, further characterized by a pH of 5 to 7.0. M13. A pharmaceutical composition comprising 5 to 25 mg / mL of an IgG:TGFβR fusion protein and 20 to 50 mM of an isotonic agent (preferably 20 to 50 mM sodium chloride), and further characterized by a pH of 5.0 to 6.5, preferably a pH of 5.0 to 5.7. M14. A pharmaceutical composition comprising 25 to 45 mg / mL of an IgG:TGFβR fusion protein and 40 to 80 mM of an isotonic agent (preferably 40 to 80 mM sodium chloride), and further characterized by a pH of 5.0 to 7.5, preferably pH 5.3 to 6.2. M15. A pharmaceutical composition comprising at least 25 mg / mL IgG:TGFβR fusion protein and at least 50 mM of a tonicity agent (preferably at least 50 mM sodium chloride), further characterized by a pH of 5.0 to 7.5, preferably a pH of 5.3 to 6.5. M16. A pharmaceutical composition comprising 45 to 65 mg / mL of an IgG:TGFβR fusion protein and 80 to 170 mM of an isotonic agent (preferably 80 to 170 mM sodium chloride), and further characterized by a pH of 5.0 to 7.5, preferably a pH of 5.5 to 6.5. M17. A pharmaceutical composition comprising 45-65 mg / mL of an IgG:TGFβR fusion protein and 80-120 mM of an isotonic agent (preferably 80-120 mM sodium chloride), further characterized by a pH of 5.0-7.5, preferably pH 5.5-6.5, and preferably also containing a sugar component (most preferably trehalose). M18. A pharmaceutical composition comprising 45-65 mg / mL of an IgG:TGFβR fusion protein and 120-170 mM of an isotonic agent (preferably 120-170 mM sodium chloride), further characterized by a pH of 5.0-7.5, preferably 5.5-6.5, and preferably in the absence of a sugar component (most preferably trehalose). M19. A pharmaceutical composition comprising 65 to 115 mg / mL of an IgG:TGFβR fusion protein and 100 to 200 mM of an isotonic agent (preferably 100 to 200 mM sodium chloride), and further characterized by a pH of 5.0 to 8.0, preferably a pH of 5.8 to 6.8. M20. Any of the pharmaceutical compositions M1 to M19, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. M21. Any of the pharmaceutical compositions M1 to M20, further comprising an isotonic agent, preferably sodium chloride. M22. The pharmaceutical composition of any one of M1 to M21, further comprising a surfactant, preferably polysorbate 20. M23. A pharmaceutical composition according to any one of M1 to M22, further comprising a sugar component, preferably trehalose. M24. A pharmaceutical composition according to any one of M1 to M23, further comprising an amino acid component, preferably arginine and / or lysine. M25. The pharmaceutical composition of any of M1 to M24, further comprising a buffer system, preferably a histidine buffer system. M26. Any of the pharmaceutical compositions M1 to M25, further comprising an antioxidant, preferably methionine. M27. The pharmaceutical composition of any of M1-M26, further characterized by the absence of acetate buffer and acetate salt. M28. Any of the pharmaceutical compositions of M1 to M27, which is a liquid pharmaceutical composition.

[0278] sugar component Experimental studies have generally shown that the inclusion of a sugar moiety, particularly a sugar moiety selected from the group consisting of non-reducing disaccharides and non-reducing sugar polyols, is advantageous, with trehalose appearing to be far superior to all other sugar moieties.

[0279] The following numbered paragraphs N1-N24 reveal specific embodiments of the present invention: N1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar moiety. N2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30 to 300 mM of a sugar component. N3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 40 to 110 mM of a sugar component. N4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 100 to 200 mM of a sugar component. N5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar moiety, the sugar moiety to fusion protein molar ratio being between 3700:1 and 70:1. N6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a sugar moiety selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol. N7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30-300 mM of a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol. N8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 40-110 mM of a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol. 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. 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, at a molar ratio of sugar component to fusion protein of 3700:1 to 70:1. N11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and trehalose. N12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30 to 300 mM trehalose. N13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 40 to 110 mM trehalose. N14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 100 to 200 mM trehalose. N15. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and trehalose at a molar ratio of trehalose to fusion protein of 3700:1 to 70:1. N16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a disaccharide. N17. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 40 to 200 mM of a disaccharide. N18. Any of the pharmaceutical compositions of N1 to N17, further characterized by any of the properties set forth in 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. N19. The pharmaceutical composition of any of N1 to N18, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. N20. Any of the pharmaceutical compositions N1 to N19, further comprising an isotonic agent, preferably sodium chloride. N21. Any of the pharmaceutical compositions N1 to N20, further comprising a surfactant, preferably polysorbate 20. N22. A pharmaceutical composition according to any one of N1 to N21, further comprising an amino acid component, preferably arginine and / or lysine. N23. ​​The pharmaceutical composition of any of N1 to N22, further comprising a buffer system, preferably a histidine buffer system. N24. Any of the pharmaceutical compositions N1 to N23, further comprising an antioxidant, preferably methionine.

[0280] Amino acid components Experimental studies have generally shown that the inclusion of amino acid components, particularly those selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine, is advantageous, with arginine, and to some extent lysine, appearing to be far superior to all other amino acid components.

[0281] The following numbered paragraphs O1-O20 reveal specific embodiments of the present invention: O1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component. O2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 10 to 300 mM of an amino acid component. O3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30 to 110 mM of an amino acid component. O4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and an amino acid component, the molar ratio of the amino acid component to the fusion protein being between 5500:1 and 18:1. 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. O6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 10-300 mM of an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof. O7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30-110 mM of an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, glycine, and any combination thereof. 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 amino acid component to fusion protein of 5500:1 to 18:1. O9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and arginine. O10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 10 to 300 mM arginine. O11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30 to 110 mM arginine. O12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 30 to 80 mM arginine. O13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and arginine, at a molar ratio of arginine to fusion protein of 5500:1 to 18:1. O14. Any of the pharmaceutical compositions O1 to O13, further characterized by any of the properties set forth in 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. O15. The pharmaceutical composition of any of O1-O14, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. O16. Any of the pharmaceutical compositions O1 to O15, further comprising an isotonic agent, preferably sodium chloride. O17. Any of the pharmaceutical compositions of O1 to O16, further comprising a surfactant, preferably polysorbate 20. O18. The pharmaceutical composition of any one of O1 to O17, further comprising a sugar component, preferably trehalose. O19. The pharmaceutical composition of any of O1 to O18, further comprising a buffer system, preferably a histidine buffer system. O20. Any of the pharmaceutical compositions O1 to O19, further comprising an antioxidant, preferably methionine.

[0282] Sugar and amino acid components Studies have shown that arginine, trehalose, and lysine all contribute to the stability of the formulation. While arginine can potentially replace or at least partially replace trehalose, the combination of trehalose and arginine has been found to provide particularly good stabilization.

[0283] The following numbered paragraphs P1 to P18 reveal specific embodiments of the present invention: P1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a sugar component, and an amino acid component. P2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 30 to 300 mM of a sugar component, and 10 to 300 mM of an amino acid component. P3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 40 to 110 mM of a sugar component, and 30 to 110 mM of an amino acid component. P4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a sugar component in a molar ratio of sugar component to fusion protein of 3700:1 to 70:1, and an amino acid component in a molar ratio of amino acid component to fusion protein of 5500:1 to 18:1. P5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 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. 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. 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. P8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a sugar component selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol in a molar ratio of the sugar component to the fusion protein of 3700:1 to 70:1, and an amino acid component selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine in a molar ratio of the amino acid component to the fusion protein of 5500:1 to 18:1. P9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose, and arginine. P10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 30 to 300 mM trehalose, and 10 to 300 mM arginine. P11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, 40-110 mM trehalose, and 30-110 mM arginine. P12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, trehalose in a molar ratio to the fusion protein of 3700:1 to 70:1, and arginine in a molar ratio to the fusion protein of 5500:1 to 18:1. P13. The pharmaceutical composition of any one of P1 to P12, further characterized by any one of the properties shown in 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. P14. The pharmaceutical composition of any of P1 to P13, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. P15. Any of the pharmaceutical compositions P1 to P14, further comprising an isotonic agent, preferably sodium chloride. P16. A pharmaceutical composition according to any one of P1 to P15, further comprising a surfactant, preferably polysorbate 20. P17. The pharmaceutical composition of any of P1 to P16, further comprising a buffer system, preferably a histidine buffer system. P18. Any of the pharmaceutical compositions P1 to P17, further comprising an antioxidant, preferably methionine.

[0284] Sugar and amino acid components as alternatives to sodium chloride Subsequent studies suggest that arginine, and to some extent trehalose, can potentially replace or partially replace NaCl as a source of ionic strength.

[0285] The following numbered paragraphs Q1-Q17 identify specific embodiments of the present invention: Q1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and one or more or all of sodium chloride, a sugar component, and an amino acid component. Q2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and one or more or all of sodium chloride and amino acid components. Q3. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of sodium chloride, a sugar component, and an amino acid component; wherein the total molar concentration of the combination of sodium chloride, the sugar component, and one or more or all of the amino acid components is 150 mM to 250 mM. Q4. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of sodium chloride, a sugar component, and an amino acid component; wherein the total molar concentration of the combination of sodium chloride, a sugar component, and one or more or all of the amino acid components is 180 mM to 220 mM. Q5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and one or more or all of the following sugar components selected from the group consisting of sodium chloride, trehalose, sucrose, mannitol, and sorbitol, and amino acid components selected from the group consisting of arginine, lysine, proline, glutamic acid, and glycine. 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. Q7. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of the following: a sugar component selected from the group consisting of sodium chloride, 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 the sodium chloride, the sugar component, and one or more or all of the amino acid components is 150 mM to 250 mM. Q8. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of the following: a sugar component selected from the group consisting of sodium chloride, 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 the sodium chloride, the sugar component, and one or more or all of the amino acid components is 180 mM to 220 mM. Q9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and one or more or all of sodium chloride, trehalose, and arginine. Q10. A pharmaceutical composition containing an IgG:TGFβR fusion protein, sodium chloride, and arginine. Q11. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of sodium chloride, trehalose, and arginine; wherein the total molar concentration of the combination of one or more or all of sodium chloride, trehalose, and arginine is 150 mM to 250 mM. Q12. A pharmaceutical composition containing an IgG:TGFβR fusion protein and one or more or all of sodium chloride, trehalose, and arginine; wherein the total molar concentration of the combination of one or more or all of sodium chloride, trehalose, and arginine is 180 mM to 220 mM. Q13. Any of the pharmaceutical compositions of Q1 to Q12, further characterized by any of the properties shown in 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. Q14. The pharmaceutical composition of any 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 that of PD-L1 alpha. Q15. Any of the pharmaceutical compositions of Q1 to Q14, further containing a surfactant, preferably polysorbate 20. Q16. Any of the pharmaceutical compositions of Q1 to Q15, further comprising a buffer system, preferably a histidine buffer system. Q17. Any of the pharmaceutical compositions of Q1 to Q16, further containing an antioxidant, preferably methionine.

[0286] surfactants Studies have shown that surfactants are desirable, with polysorbate surfactants performing best, particularly polysorbate 20. Surfactants have been particularly useful in reducing aggregation.

[0287] The following numbered paragraphs R1-R34 define specific embodiments of the present invention: R1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant. R2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.01 to 2 mg / mL of a surfactant. R3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.05 to 1.5 mg / mL of a surfactant. R4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 1.2 mg / mL of a surfactant. R5. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 0.6 mg / mL of a surfactant. R6. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and about 0.5 mg / mL of a surfactant. R7. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant at a molar ratio of surfactant to fusion protein of 30:1 to 1:70. R8. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant at a molar ratio of surfactant to fusion protein of 12:1 to 1:3. R9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant at a molar ratio of surfactant to fusion protein of 8:1 to 1:2. R10. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80. R11. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.01 to 2 mg / mL of a surfactant selected from polysorbate 20 and polysorbate 80. R12. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.05 to 1.5 mg / mL of a surfactant selected from polysorbate 20 and polysorbate 80. R13. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 1.2 mg / mL of a surfactant selected from polysorbate 20 and polysorbate 80. R14. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 0.6 mg / mL of a surfactant selected from polysorbate 20 and polysorbate 80. 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. R16. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80, at a molar ratio of surfactant to fusion protein of 30:1 to 1:70. 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 surfactant to fusion protein of 12:1 to 1:3. R18. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a surfactant selected from polysorbate 20 or polysorbate 80, at a molar ratio of surfactant to fusion protein of 8:1 to 1:2. R19. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and polysorbate 20. R20. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.01 to 2 mg / mL of polysorbate 20. R21. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.05 to 1.5 mg / mL of polysorbate 20. R22. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 1.2 mg / mL of polysorbate 20. R23. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and 0.4 to 0.6 mg / mL of polysorbate 20. R24. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and about 0.5 mg / mL polysorbate 20. R25. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and polysorbate 20 at a molar ratio of polysorbate 20 to the fusion protein of 30:1 to 1:70. R26. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and polysorbate 20 in a molar ratio of polysorbate 20 to the fusion protein of 12:1 to 1:3. R27. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and polysorbate 20 at a molar ratio of polysorbate 20 to the fusion protein of 8:1 to 1:2. R28. Any of the pharmaceutical compositions R1 to R27, further characterized by any of the properties shown in 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. R29. The pharmaceutical composition of any of R1 to R28, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. R30. A pharmaceutical composition according to any one of R1 to R29, further comprising an isotonic agent, preferably sodium chloride. R31. The pharmaceutical composition of any one of R1 to R30, further comprising a sugar component, preferably trehalose. R32. A pharmaceutical composition according to any one of R1 to R31, further comprising an amino acid component, preferably arginine and / or lysine. R33. The pharmaceutical composition of any of R1 to R32, further comprising a buffer system, preferably a histidine buffer system. R34. The pharmaceutical composition of any of R1 to R33, further comprising an antioxidant, preferably methionine.

[0288] buffer solution It is preferred that the pharmaceutical compositions of the present invention contain a buffer system, as compositions without a buffer appear to be less preferred than those containing a buffer. A number of buffer systems may be used to buffer the pharmaceutical compositions of the present invention at an appropriate pH (preferably as defined herein), although some buffers appear to be more preferred than others. However, studies have shown that the buffer concentration does not appear to be very important.

[0289] Generally, histidine buffer system performs best.Acetate buffer can be used, but it is preferably excluded due to the possibility of skin irritation.Similarly, citrate buffer can be useful at relatively high pH, ​​but citrate buffer is also advantageously absent from the pharmaceutical composition of the present invention.

[0290] The following numbered paragraphs S1-S18 reveal specific embodiments of the present invention: S1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system. S2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system selected from the group consisting of a histidine buffer, a phosphate buffer, a succinate buffer, a citrate buffer, and any combination thereof. S3. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a buffer system selected from the group consisting of a histidine buffer, a phosphate buffer, a succinate buffer, and a citrate buffer. S4. A pharmaceutical composition comprising an IgG:TGFβR fusion protein and a histidine buffer system. S5. The pharmaceutical composition of any of S1 to S4, wherein the buffer system is present at a concentration of 2 to 70 mM. S6. The pharmaceutical composition of any of S1 to S4, wherein the buffer system is present at a concentration of 4 to 30 mM. S7. The pharmaceutical composition of any of S1 to S4, wherein the buffer system is present at a concentration of 5 to 15 mM. S8. The pharmaceutical composition of any of S1-S4, wherein the buffer system is present in a molar ratio of buffer system to fusion protein of 1280:1 to 3:1. S9. The pharmaceutical composition of any of S1-S4, wherein the buffer system is present in a molar ratio of buffer system to fusion protein of 370:1 to 9:1. S10. The pharmaceutical composition of any of S1-S4, wherein the buffer system is present in a molar ratio of buffer system to fusion protein of 100:1 to 15:1. S11. The pharmaceutical composition of any of S1-S4, wherein the composition is further characterized by the absence of acetate buffer and acetate salt. S12. Any of the pharmaceutical compositions S1 to S11, further characterized by any of the properties shown in 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. S13. The pharmaceutical composition of any of S1 to S12, wherein the IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR2 fusion protein, preferably having an amino acid sequence identical to the amino acid sequence of PD-L1 alpha. S14. The pharmaceutical composition of any one of S1 to S13, further comprising an isotonic agent, preferably sodium chloride. S15. The pharmaceutical composition of any one of S1 to S14, further comprising a surfactant, preferably polysorbate 20. S16. A pharmaceutical composition according to any one of S1 to S15, further comprising a sugar component, preferably trehalose. S17. The pharmaceutical composition of any one of S1 to S16, further comprising an amino acid component, preferably arginine and / or lysine. S18. The pharmaceutical composition of any one of S1 to S17, further comprising an antioxidant, preferably methionine.

[0291] In view of experimental studies, the following pharmaceutical compositions disclosed in T1 to T19 have been found to be particularly stable: T1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a buffer system, NaCl; a sugar component, a non-ionic surfactant, and optionally an amino acid antioxidant, characterized by a pH of 5.0 to 6.0. T2. The pharmaceutical composition of T1, comprising 2-30 mg / mL of IgG:TGFβR fusion protein, 2-30 mM of a buffer system, 20-70 mM NaCl; 100-200 mM of a sugar component, 0.1-1.1 mg / mL of a non-ionic surfactant, and optionally 1-20 mM of an amino acid antioxidant, and characterized by a pH of 5.0-6.0. T3. The pharmaceutical composition of T1, comprising 5-15 mg / mL of IgG:TGFβR fusion protein, 5-15 mM of a buffer system, 30-50 mM NaCl; 150-170 mM of a sugar component, 0.3-0.7 mg / mL of a non-ionic surfactant, and optionally 2-8 mM of an amino acid antioxidant, and characterized by a pH of 5.3-5.7. T4. The pharmaceutical composition of 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 non-ionic surfactant, and optionally about 5 mM amino acid antioxidant, and characterized by a pH of about 5.5. T5. The pharmaceutical composition of 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; and characterized by a pH of 5.0-6.0. T6. The pharmaceutical composition of T1, comprising 35-45 mg / mL IgG:TGFβR fusion protein, 5-15 mM buffer system, 50-70 mM NaCl; 150-170 mM sugar component, 0.3-0.7 mg / mL non-ionic surfactant, and optionally 2-8 mM amino acid antioxidant; and characterized by a pH of 5.3-5.7. T7. The pharmaceutical composition of 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 non-ionic surfactant, and optionally about 5 mM amino acid antioxidant, and characterized by a pH of about 5.5. T8. Any of the pharmaceutical compositions T1 to T7, wherein the IgG:TGFβR fusion protein has an amino acid sequence identical to that of bintrafus alpha, 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. T9. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, a buffer system, NaCl; a sugar component, an amino acid component, a non-ionic surfactant, and optionally an amino acid antioxidant, and characterized by a pH of 5.4 to 6.4. T10. The pharmaceutical composition of 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 non-ionic surfactant, and optionally 1-20 mM amino acid antioxidant, and characterized by a pH of 5.4-6.4. T11. The pharmaceutical composition of T9, comprising 35-45 mg / mL IgG:TGFβR fusion protein, 5-15 mM buffer system, 50-70 mM NaCl; 90-110 mM sugar component, 40-60 mM amino acid component, 0.3-0.7 mg / mL non-ionic surfactant, and optionally 2-8 mM amino acid antioxidant; and characterized by a pH of 5.7-6.1. T12. The pharmaceutical composition of 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 non-ionic surfactant, and optionally about 5 mM amino acid antioxidant, and characterized by a pH of about 5.9. T13. The pharmaceutical composition of T9, comprising 25-100 mg / mL of IgG:TGFβR fusion protein, 2-30 mM of a buffer system, 40-100 mM NaCl; 25-125 mM of a sugar component, 25-125 mM of an amino acid component, 0.1-1.1 mg / mL of a non-ionic surfactant, and optionally 1-20 mM of an amino acid antioxidant; and characterized by a pH of 5.4-6.4. T14. The pharmaceutical composition of T9, comprising 35-45 mg / mL IgG:TGFβR fusion protein, 5-15 mM buffer system, 50-70 mM NaCl; 65-85 mM sugar component, 65-85 mM amino acid component, 0.3-0.7 mg / mL non-ionic surfactant, and optionally 2-8 mM amino acid antioxidant; and characterized by a pH of 5.7-6.1. T15. The pharmaceutical composition of 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 non-ionic surfactant, and optionally about 5 mM amino acid antioxidant, and characterized by a pH of about 5.9. T16. The pharmaceutical composition of T9, comprising 35-120 mg / mL IgG:TGFβR fusion protein, 2-30 mM buffer system, 50-150 mM NaCl; 10-100 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; and characterized by a pH of 5.4-6.4. T17. The pharmaceutical composition of T9, comprising 45-55 mg / mL IgG:TGFβR fusion protein, 5-15 mM buffer system, 90-110 mM NaCl; 40-60 mM sugar component, 40-60 mM amino acid component, 0.3-0.7 mg / mL non-ionic surfactant, and optionally 2-8 mM amino acid antioxidant; and characterized by a pH of 5.7-6.1. T18. The pharmaceutical composition of 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 non-ionic surfactant, and optionally about 5 mM amino acid antioxidant, and characterized by a pH of about 5.9. T19. Any of the pharmaceutical compositions T1 to T18, wherein the IgG:TGFβR fusion protein has an amino acid sequence identical to that of bintrafus alpha, 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.

[0292] In addition to those mentioned above, the following embodiments U1 to U21 are particularly preferred: U1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an ionic tonicity agent, and one or both of a disaccharide and an amino acid component. U2. The pharmaceutical composition of U1, wherein the composition comprises a disaccharide. U3. The pharmaceutical composition of U1 or U2, wherein the ionic tonicity agent is sodium chloride. U4. The pharmaceutical composition of any one of U1 to U3, wherein the disaccharide is trehalose or sucrose. U5. The pharmaceutical composition of any one of U1 to U4, wherein the disaccharide is trehalose. U6. The pharmaceutical composition of any one of U1 to U5, wherein the amino acid component is arginine or lysine. U7. The pharmaceutical composition of any one of U1 to U6, wherein the amino acid component is arginine. U8. The pharmaceutical composition of any one of U1 to U7, wherein the ionic tonicity agent is sodium chloride, the disaccharide is trehalose, and the amino acid component is arginine. U9. Any one of the pharmaceutical compositions U1 to U8, further comprising a surfactant. U10. Any one of the pharmaceutical compositions U1 to U9, wherein the composition has a pH of 5.0 to 6.5. U11. Any one of the pharmaceutical compositions 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-L1(IgG):TGFβR fusion protein. U12. The IgG:TGFβR fusion protein is an anti-PD-L1(IgG):TGFβR fusion protein, and the anti-PD-L1(IgG) is (1) an anti-PD-L1(IgG) comprising three heavy chain CDRs having the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2), and SEQ ID NO:21 (CDR3), and three light chain CDRs having the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2), and SEQ ID NO:24 (CDR3); (2) an anti-PD-L1(IgG) comprising three heavy chain CDRs having the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3); and three light chain CDRs having the amino acid sequences of SEQ ID NO: 30 (CDR1), SEQ ID NO: 31 (CDR2), and SEQ ID NO: 32 (CDR3). U13. The pharmaceutical composition of U12, wherein the anti-PD-L1 (IgG) light chain and heavy chain sequences 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. 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 to TGF-β. U15. The pharmaceutical composition of U14, wherein the TGFβR2 comprises or consists of a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. U16. Any one of the pharmaceutical compositions U1 to U15, wherein the light chain and heavy chain sequences 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. U17. The pharmaceutical composition of any one of U1 to U16, wherein the IgG:TGFβR fusion protein has the amino acid sequence of vintrofs alpha. U18. Any one of the pharmaceutical compositions of U1 to U17, wherein the composition is characterized by a pH of 5.0 to 6.5 and contains a buffer system, an ionic tonicity agent, a disaccharide, a non-ionic surfactant, optionally an amino acid component, and further optionally an antioxidant. U19. The pharmaceutical composition of U18, wherein the buffer system is a histidine buffer system, the ionic tonicity agent is sodium chloride, the disaccharide is trehalose, the amino acid component is arginine, the antioxidant is methionine, and the non-ionic surfactant is polysorbate 20. U20. The pharmaceutical composition of U18 or U19, wherein the composition is selected from the group of compositions: a. A pharmaceutical composition characterized by a pH of 5.3 to 5.7 and containing 5 to 15 mg / mL of an IgG:TGFβR fusion protein, 5 to 15 mM of a buffer system, 30 to 50 mM of an ionic tonicity agent; 150 to 170 mM of a disaccharide, 0.3 to 0.7 mg / mL of a non-ionic surfactant, and optionally 2 to 8 mM of an antioxidant; b. A pharmaceutical composition characterized by a pH of 5.3 to 5.7 and containing 35 to 45 mg / mL of an IgG:TGFβR fusion protein, 5 to 15 mM of a buffer system, 50 to 70 mM of an ionic tonicity agent; 150 to 170 mM of a disaccharide, 0.3 to 0.7 mg / mL of a non-ionic surfactant, and optionally 2 to 8 mM of an antioxidant; c. A pharmaceutical composition characterized by a pH of 5.7 to 6.1 and containing 35 to 45 mg / mL of an IgG:TGFβR fusion protein, 5 to 15 mM of a buffer system, 50 to 70 mM of an ionic tonicity agent; 90 to 110 mM of a disaccharide, 40 to 60 mM of an amino acid component, 0.3 to 0.7 mg / mL of a non-ionic surfactant, and optionally 2 to 8 mM of an antioxidant; d. A pharmaceutical composition characterized by a pH of 5.7-6.1 and containing 35-45 mg / mL of an IgG:TGFβR fusion protein, 5-15 mM of a buffer system, 50-70 mM of an ionic tonicity agent; 65-85 mM of a disaccharide, 65-85 mM of an amino acid component, 0.3-0.7 mg / mL of a non-ionic surfactant, and optionally 2-8 mM of an antioxidant; and e. A pharmaceutical composition characterized by a pH of 5.7 to 6.1 and containing 45 to 55 mg / mL of IgG:TGFβR fusion protein, 5 to 15 mM of a buffer system, 90 to 110 mM of an ionic tonicity agent; 40 to 60 mM of a disaccharide, 40 to 60 mM of an amino acid component, 0.3 to 0.7 mg / mL of a non-ionic surfactant, and optionally 2 to 8 mM of an antioxidant. U21. The pharmaceutical composition of any one of U1-U20, wherein the ionic tonicity agent is a non-buffering ionic tonicity agent.

[0293] container The present invention provides a container comprising a pharmaceutical composition as defined herein, preferably comprising a drug delivery device as defined herein, preferably a plurality of drug delivery devices.

[0294] The present invention provides a method of manufacturing a container, the method comprising incorporating a pharmaceutical composition as defined herein into the container, preferably 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.

[0295] Drug Delivery Devices The present invention provides a drug delivery device comprising a pharmaceutical composition as defined herein. Preferably, the drug delivery device comprises a chamber in which the pharmaceutical composition, preferably a liquid (most preferably aqueous), is present. Preferably, the drug delivery device is sterilized.

[0296] The drug delivery device may be a vial, an ampoule, a syringe, an injection pen (e.g., essentially incorporating a syringe), an autoinjector, or an intravenous bag. Most preferably, the drug delivery device is a syringe. Preferably, the syringe is a glass syringe. Preferably, the syringe includes a needle.

[0297] The present invention provides a method of manufacturing a drug delivery device, preferably as defined herein, which method comprises incorporating a pharmaceutical composition, as defined herein, into the drug delivery device. Such manufacturing typically involves loading a pharmaceutical composition, as defined herein, into a syringe, preferably via a needle attached thereto. The needle may then be removed, replaced, or left in place.

[0298] Parts kit The present invention provides a kit of parts comprising a drug delivery device (without a pharmaceutical composition incorporated therein), a pharmaceutical composition as defined herein (optionally contained in a separate package or container), and optionally a set of instructions for administering the pharmaceutical composition (e.g., intravenously, subcutaneously). The user then loads the drug delivery device with the pharmaceutical composition (which may be provided in a vial, ampoule, etc.) prior to administration.

[0299] Manufacturing method The present invention provides a method for preparing a pharmaceutical composition as defined herein. This method preferably comprises mixing together, in any particular order deemed appropriate, any relevant components required to form the composition defined herein. Those skilled in the art can refer to the examples or techniques well known in the art for forming pharmaceutical compositions (particularly those for injection by syringe, and particularly those for intravenous injection). Various embodiments will preferably require different combinations of components to be mixed, potentially in different amounts. Those skilled in the art can readily deduce such combinations and amounts by reference to the above disclosure regarding the pharmaceutical composition.

[0300] The method preferably involves mixing the relevant ingredients together, preferably in a diluent (e.g., water), such that all ingredients are preferably dissolved (substantially or completely) in the diluent.

[0301] The method may involve first preparing a concentrated pre-mixture (or pre-solution) of some or all of the ingredients (optionally with some or all of the diluent) including the fusion protein, and then diluting the pre-mixture (or pre-solution) with a diluent, preferably water, to provide a pharmaceutical composition, or a composition to which the final ingredient is subsequently added to provide the final pharmaceutical composition. Preferably, the pre-mixture is prepared at the desired pH for the final formulation.

[0302] The method may alternatively or additionally involve first preparing a pre-mixture (or pre-solution) of some or all of the components except the fusion protein (optionally with some or all of the diluent), and then mixing the fusion protein by itself (optionally with or pre-dissolved in some of the diluent) with the pre-mixture (or pre-solution) to provide a pharmaceutical composition, or a composition to which the final components are then added to provide the final pharmaceutical composition. Preferably, this pre-mixture contains all of the components except the fusion protein, and optionally also some of the diluent (which may be used to pre-dissolve the fusion protein), such that the fusion protein is added to a mixture that provides optimal stabilization of the fusion protein. Preferably, the pre-mixture is prepared at the desired pH for the final formulation.

[0303] Preferably, the method involves forming a buffer system, preferably a buffer system containing a buffering agent as defined herein. The buffer system may have a pH, preferably as defined herein, relative to the buffer system itself. The buffer system is preferably formed in a pre-mixture prior to addition of the fusion protein, although the buffer system may optionally be formed by the fusion protein present. The buffer system may be formed by simply mixing the buffering agent (supplied ready-made) with its acid / base conjugate (preferably in the appropriate relative amounts to provide the desired pH—this can be determined by one of ordinary skill in the art either theoretically or experimentally). In the case of an acetate buffer system, this means mixing sodium acetate with acetic acid. Alternatively, the buffer system may be formed through the addition of a strong acid (e.g., HCl) to a buffering agent (e.g., sodium acetate) to form an acid / base conjugate (e.g., acetic acid) in situ (again, preferably in the appropriate relative amounts to provide the desired pH). Alternatively, the buffer system may be formed through the addition of a strong base (e.g., sodium hydroxide) to an acid / base conjugate (e.g., acetic acid) of a buffering agent (e.g., sodium acetate) to form a buffer in situ (again, preferably in the appropriate relative amounts to provide the desired pH). The pH of any pre-mixture of the final composition may be adjusted with discretion by adding the required amount of strong base or strong acid, or even an amount of buffering agent or acid / base conjugate. The same protocol may be used in connection with other buffer systems, such as a histidine buffer system, which may be formed using either a combination of free (neutral) histidine with the imidazolium form of histidine, or subsequent adjustment of pH (e.g., with the required base or acid).

[0304] In some embodiments, the buffer and / or buffer system is pre-formed as a separate mixture, and the buffer system is transferred to the precursor of the composition (containing some or all of the components that contribute to the buffer and / or buffer system, preferably containing the fusion protein) via buffer exchange (e.g., using diafiltration until the relative concentration or osmolality is reached). Additional excipients can then be added if necessary to create the final composition. The pH can be adjusted once or before all components are present.

[0305] Any, some, or all of the ingredients may be pre-dissolved or pre-mixed with a diluent before mixing with the other ingredients.

[0306] The final composition may be filtered, preferably to remove particulate matter. Preferably, the filtration is through a filter of 1 μm or less, preferably 0.22 μm size. Preferably, the filtration is through either a PES or PVDF filter, preferably a 0.22 μm PES filter.

[0307] It will be understood that the pharmaceutical compositions of the present invention are provided as liquid (preferably aqueous) pharmaceutical compositions and are preferably stored duly. However, the pharmaceutical compositions of the present invention may also be provided as lyophilized pharmaceutical compositions and preferably stored duly. Such lyophilized pharmaceutical compositions may be formed by freeze-drying a liquid (e.g., aqueous) pharmaceutical composition as defined herein. Most preferably, however, any such lyophilized pharmaceutical composition is reconstituted prior to use, administration, or even prior to (potentially short-term) storage to provide a liquid or aqueous pharmaceutical composition as defined herein. Preferably, such reconstitution comprises dissolving the lyophilized composition in water (for injection), preferably to provide a solution of the fusion protein at the desired concentration.

[0308] Treatment method The present invention provides methods for treating diseases or medical disorders by administering a pharmaceutical composition as defined herein. Such methods preferably comprise administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof. Administration preferably includes parenteral administration, including any form of administration other than enteral, and local administration, usually by injection, and preferably includes, but is not limited to, intravenous, intravitreal, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Such methods preferably comprise subcutaneous or intravenous delivery of a therapeutically effective amount of the pharmaceutical composition. Such methods preferably comprise intravenous administration of a therapeutically effective amount of the pharmaceutical composition, preferably by intravenous injection, preferably together with other intravenous fluids (e.g., saline).

[0309] If the pharmaceutical composition is a lyophilized pharmaceutical composition, prior to administration, the composition is preferably reconstituted in water for injection or a suitable intravenous fluid, preferably to provide an aqueous pharmaceutical composition as defined herein, which is then preferably administered as defined herein, preferably together with other intravenous fluids (e.g., saline), preferably by intravenous injection.

[0310] The invention provides a pharmaceutical composition as defined herein for use in the treatment of a disease or medical disorder in a patient in need of such treatment. The invention also provides the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a disease or disorder.

[0311] The present invention provides methods of treating a disease or medical disorder, pharmaceutical compositions for use in treating a disease or medical disorder, and the use of a pharmaceutical composition in the manufacture of a medicament for treating a disease or disorder, as defined herein, wherein the disease or medical disorder is a PD-L1- and / or TGFβ-associated disease, preferably a PD-L1- and / or TGFβ-associated proliferative disorder, more preferably a PD-L1- and / or TGFβ-associated cancer. In such aspects of the invention, the anti-PD-L1 antigen-binding portion of the anti-PD-L1(IgG):TGFβR fusion protein binds to tumor-borne PD-L1, thereby disrupting the PD-1 / PD-L1 pathway, while localizing to the TGFβ receptor portion of the fusion protein within the tumor microenvironment, where it can most effectively target and / or capture autocrine or paracrine TGFβ. In such a manner, multiple mechanisms of action are disrupted, thereby blocking growth and / or survival pathways of cancer cells.

[0312] The present invention provides methods of treating a disease or medical disorder, pharmaceutical compositions for use in treating a disease or medical disorder, and the use of pharmaceutical compositions in the manufacture of a medicament for treating a disease or disorder, as defined herein, wherein the disease or medical disorder is a proliferative disease or disorder. The proliferative disease or disorder is preferably cancer. The cancer is preferably manifested by one or more solid tumors. The cancer is preferably selected from carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tract) cancer, renal 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 tumor, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, bile duct cancer, and head and neck cancer. The disease or medical disorder in question 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.

[0313] The present invention provides a method for treating a disease or medical disorder as defined herein, a pharmaceutical composition for use in treating a disease or medical disorder, and the use of a pharmaceutical composition in the manufacture of a medicament for treating a disease or disorder, wherein the disease or medical disorder is preferably as defined herein, and wherein the treatment or treatment involves a combination therapy, whereby the pharmaceutical composition is administered in combination with one or more other pharmaceutically or biopharmaceutical active substances (may be multiple); wherein the combination therapy involves simultaneous, sequential, or separate administration of the individual components of this treatment.In some embodiments, additional pharmaceutically or biopharmaceutical active substances can be present in any of the pharmaceutical compositions as defined herein.

[0314] All references cited herein are hereby incorporated by reference in this disclosure. [Example]

[0315] Example Anti-PD-L1 Bioactivity Assay Protocol The biological activity of the anti-PD-L1(IgG):TGFβRII fusion protein was assessed using a cell-based assay that measured its ability to bind to the human PD-L1 receptor overexpressed on a recombinant HEK-293(hPD-L1) cell line. To this end, the anti-PD-L1(IgG):TGFβRII fusion protein was bound to a Protein A-coated 96-well plate for 30 minutes at room temperature with swirling. 50,000 HEK-293(hPD-L1) cells were then added to each well of the anti-PD-L1(IgG):TGFβRII-coated plate and allowed to bind to the anti-PD-L1(IgG):TGFβRII for 1 hour at 37°C, 5% CO2. Unbound cells were washed away, and the presence of bound cells in each well was confirmed using ATPlite 1 step (Perkin Elmer). The counts / second were then plotted against the log-transformed anti-PD-L1(IgG):TGFβRII concentration and fitted by 4PL (sigmoidal dose-response curve). For each data set, the concentration of anti-PD-L1(IgG):TGFβRII capable of binding 50% of the maximum possible PDL-1 binding capacity (EC50) was calculated. The biological activity of the samples was expressed as % activity relative to the reference substance and is expressed as a percentage of the potency ratio (i.e., the EC50 ratio of equipotent doses of the standard and unknown preparations). The potency is the average result from three independent assays.

[0316] TGFβRII Bioactivity Assay Protocol The TGFβ-binding activity of the anti-PD-L1(IgG):TGFβRII fusion protein was assessed using the reporter cell line 4T1 pSmad sLuc, which is stably transfected with a luciferase reporter gene controlled by an SMAD-binding element. Upon binding of TGFβ to the TGFβ receptor on the cell surface of this cell line, a signaling cascade is initiated that results in the formation of a SMAD complex. This SMAD complex then translocates to the nucleus, where it binds to the SMAD-binding element and induces transcription of the luciferase gene.

[0317] For each bioassay, 50,000 4T1 cells per well were added to a 96-well plate and allowed to adhere to the plate 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 so that dose-response curves could be generated. Luciferase expression was then assessed using a luciferin-based detection dye. Because the anti-PD-L1(IgG):TGFβRII fusion protein captures and neutralizes soluble TGFβ, luminescence was inversely correlated with the TGFβ-binding activity of the anti-PD-L1(IgG):TGFβRII fusion protein.

[0318] For each data set, the concentration of anti-PD-L1(IgG):TGFβRII capable of binding 50% of the maximum possible TGFβ binding capacity (EC50) was calculated. Sample biological activity is expressed as % activity relative to the reference substance and is expressed as a percentage of the potency ratio (i.e., the EC50 ratio of equipotent doses of the standard and unknown preparations). The potency is the average result from three independent assays.

[0319] Stress Test Protocol The following stress testing protocol is used in the examples below, variations of which will be apparent from the context.

[0320] temperature stability The temperature stability of the formulations was tested over a storage period (e.g., 8, 9, or 13 weeks) at 40±2°C (75% RH). Samples were analyzed not only at time=0 and time=full period (e.g., 8 weeks), but also at intermediate intervals (e.g., time=2 weeks, time=4 weeks). Occasionally, back-up samples were placed at 25±5°C for similar periods to allow for the analysis of samples that degraded too quickly or otherwise produced unusual results at 40±2°C. Temperature stability samples were generally analyzed for: Protein content by optical density Aggregation Index: calculated by optical density to track the formation of aggregates and HMW impurities HMW content by SE-UPLC (following HMW impurity formation and thus monitoring aggregation) Turbidity by nephelometry (also related to solubility and aggregate formation) Visual inspection for the presence of visible particles and / or changes in the degree of coloration Charge isoforms by isoelectric focusing capillary electrophoresis (to monitor charge variants) pH LMW content by bioanalyzer or CGE-non-reducing (to track fragmentation) Invisible particles due to light obstruction · Fluorescence (selected samples): To monitor changes in the three-dimensional structure of molecules.

[0321] Light stress The photosensitivity of the IgG:TGFβR2 fusion protein in various formulations was also investigated. The formulations were generally exposed to light at an intensity of 765 W / m 2 The light-stressed formulations were generally analyzed by the following techniques: Protein content by optical density Aggregation index: Calculated by OD, measures the degree of aggregate formation resulting from light stress Turbidity measured by nephelometry Visual inspection: for the presence of visible particles resulting from agglomeration SE-UPLC: Quantification of HMW impurities arising from aggregation Charge isoforms by isoelectric focusing capillary electrophoresis (to monitor charge variants) Fluorescence (selected samples): To observe any changes in the three-dimensional structure that may result from oxidation of aromatic amino acid side chains.

[0322] Shaking stress Mechanical (shaking) stress is often associated with protein self-association and the formation of aggregates due to interactions between hydrophobic regions of proteins in solution. Various formulations of IgG:TGFβR2 fusion protein were tested for resistance to shaking stress, typically at 200 mg / vial and 600 mg / vial, in both vertical and oblique positions, agitated at 200 rpm (or 300 rpm) for 24 hours at room temperature. Shaking-stressed formulations were generally analyzed as follows: Protein content by optical density Agglomeration index: calculated by optical density to track the formation of agglomeration and HMW impurities Turbidity by nephelometry (also related to solubility and aggregate formation) Visual inspection for visible particles and / or degree of coloration HMW content by SE-UPLC (following HMW impurity formation and thus monitoring aggregation) LMW content by bioanalyzer or CGE-non-reducing (to track fragmentation) -Particles not visible by light obscuration method pH.

[0323] Freeze / thaw stress When a protein formulation freezes, interfaces form as microdomains within the solidifying solution. As different components of the formulation buffer are excluded from or included in this solidifying liquid matrix, there are changes in polarity in these microenvironments. Hydrophilic / hydrophobic interactions are forced on molecules within these changing microenvironments, resulting in protein precipitation. To elucidate the effectiveness of various excipients and conditions, various formulations of IgG:TGFβR2 fusion protein were subjected to three freeze-thaw cycles (-25°C to RT each cycle), typically with 200 mg / vial or 600 mg / vial samples. These samples were then generally tested by the following assays to determine their resistance to freeze-thaw precipitation / aggregation / degradation: Protein content by optical density Agglomeration index: calculated by optical density to track the formation of agglomeration and HMW impurities Charge isoforms by isoelectric focusing capillary electrophoresis (to monitor charge variants) Turbidity by nephelometry (also related to solubility and aggregate formation) Visual inspection for the presence of visible particles HMW content by SE-UPLC (following HMW impurity formation and thus monitoring aggregation) LMW content by bioanalyzer or CGE-non-reducing (to track fragmentation) Invisible particles pH.

[0324] Results analysis and data handling protocol A partial design of experiments (DoE) was established using the "response surface model" option in Design-Expert software, with a D-Optimal Design involving three numerical factors (buffer strength, pH, surfactant strength) and one categorical factor (excipient type). To determine whether a positive response to a given stress existed, the data were statistically evaluated by ANOVA using a "response surface linear model." Using specialized design software, the relationships between all variables in this DoE were investigated, and the results were illustrated in a 3D surface plot, depicting factors that significantly contributed to the response to a given stress. In this way, a statistically significant association could be established between a given factor and the response to stress.

[0325] Formulation Preparation Protocol VintrafusAlpha drug substance (an example of an IgG:TGFβR2 fusion protein) can be produced as shown in WO2015118175, the Examples of which describe its production. The complete heavy chain of IgG connected to the TGFβR2 extracellular domain via a linker is shown as 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 VintrafusAlpha corresponds to SEQ ID NO:1 of WO2015118175.

[0326] Figure 1A shows the light chain (V) connected to the extracellular domain (ECD) of TGFβ receptor II via the indicated linker. L and C L region) and heavy chain (V H , C H 1. C H 2, and C H 3 shows half of the bintrafsalpha fusion protein, containing the nucleotide sequence of ...

[0327] Figure 1B shows the light chain (V) connected to the extracellular domain (ECD) of TGFβ receptor II via the indicated linker. L and C Lregion) and heavy chain (V H , C H 1. C H 2, and C H 1A and 1B show the sequence of half of the bintrofsalpha fusion protein, which contains the nucleotide sequence of ...

[0328] Purified bintrafus alpha was stored in a pre-formulated state at -70°C and thawed before use.

[0329] In one method, aqueous formulations were prepared by mixing a pre-prepared excipient-containing solution (at a specified pH, if applicable) with an appropriate weighed amount of Bottle Trough Alpha to provide the desired formulation in terms of Bottle Trough Alpha and excipient concentrations. A final pH adjustment was optionally made to ensure the desired pH. In another method, aqueous formulations can be made by dissolving or mixing Bottle Trough Alpha in water, optionally containing a portion of the excipients, followed by adding additional excipients (optionally pre-dissolved in water), providing the final desired formulation—again making a final pH adjustment.

[0330] In some methods, stock solutions of bintrofus alpha were made (e.g., by the methods described above) and optionally stored at low temperatures prior to use / processing. Such stock solutions were then processed, e.g., by performing buffer exchange, concentration, dilution, etc., to provide the desired formulation.

[0331] In the Examples below, it will be clear to one skilled in the art how to prepare the relevant formulations.

[0332] Example 1 - Lyophilized and liquid formulations of IgG:TGFβR2 fusion protein General Overview Preparing stable formulations for IgG:TGFβR2 fusion proteins is particularly challenging, in part because the two fused entities, the IgG portion and the TGFβR2 portion, are poorly matched with respect to pI and colloidal stability - the pI of the TGFβR2 region is radically different from that of the IgG portion, and furthermore, it is generally recognized that it is important to adopt a pH that is at least 1-2 pH units away from the pI of each major protein portion.

[0333] List of tested formulations An initial screen was performed to establish a workable and optimal pH, focusing on colloidal and conformational stability. The formulations for this initial screen are shown in Table 1A. [Table 1A]

[0334] Further experiments were performed to establish the effect of sodium chloride on solubility and dialysis recovery. These formulations are shown in Table 1B. [Table 1B]

[0335] To evaluate lyophilization performance, further screens for freeze-drying capacity and osmolality were performed on 18 formulations containing various concentrations of sodium chloride and trehalose (wt% trehalose amounts are relative to wt% trehalose dihydrate) at pH 5.5. The API was excluded from these formulations because it had little impact on the factor under observation (lyophilizate appearance) due to its negligible effect on osmolality. Therefore, such placebo formulations can be considered the worst case in terms of lyophilizate appearance. These formulations are shown in Table 1C. [Table 1C]

[0336] Further screening for freeze-dryability and osmolality was then performed on six substantially isotonic formulations containing precise concentrations of sodium chloride and trehalose at pH 5.5. These formulations are shown in Table 1D. Again, APIs were excluded for the same reasons as in Table 1C. [Table 1D]

[0337] Further screening was then performed on two substantially isotonic formulations containing 10 mg / mL Bottlelafus Alpha and precise concentrations of sodium chloride and trehalose, pH 5.5. These formulations are shown in Table 1E. [Table 1E]

[0338] 1A Formulation - Results and Analysis Nano-DSC measurements were performed on formulations 1A.1-1A.8 (see Table 1A above) to determine the conformational stability of these formulations, and the results are presented in Table 1F below. [Table 1F]

[0339] The Tm measurement results suggest that the optimum pH from the viewpoint of conformational stability is between 5.5 and 9. Furthermore, the addition of NaCl is not detrimental, but does not improve conformational stability by itself. At pH 4 and 5, without NaCl, the Tm value is slightly decreased, suggesting a decrease in conformational stability.

[0340] 1B Formulation - Results and Analysis The formulations in Table 1B above (Formulations 1B.1-1B.18) were subjected to a solubility assay measuring % protein recovery after reformulation / rebuffering to determine the colloidal stability of these formulations. The results are presented in Table 1G below. [Table 1G]

[0341] Overall, pH 5.5 showed a better solubility profile at concentrations between 1 and 10 mg / mL than pH 7. However, NaCl had a significant effect on % protein recovery—this was especially pronounced at higher pHs.

[0342] 1C Formulation - Results and Analysis The formulations in Table 1C (Formulations 1C.1 to 1C.18), all of which have a pH of 5.5 and contain 10 mg / mL bottle trough alpha, were tested in the freeze-dried state (via standard freeze-thaw cycles) and, when composed of 10 mg / mL bottle trough alpha, also for osmolality (using freezing point depression methods commonly employed in the pharmaceutical industry).

[0343] The results are shown in Table 1H below. [Table 1H]

[0344] Figure 2 shows a graphical representation of the relationship between Tg' (°C) and NaCl concentration as determined by nano-differential scanning calorimetry for formulations containing 8% trehalose (diamonds), 4% trehalose (squares), and 2% trehalose (triangles). From a practical standpoint during freeze-drying, Tg' values ​​below -40°C are undesirable.

[0345] This data indicates that there is a promising window at approximately 40 mM NaCl in terms of balancing freeze-drying feasibility and osmolality (i.e., a substantially isotonic formulation). Furthermore, trehalose concentrations of 4% to 6% appear desirable from a freeze-drying perspective. More generally, molar ratios of isotonic cryoprotectant to ionic tonicity agent of 10:1 to 1:2, more preferably 5:1 to 2:1, appear particularly suitable with respect to freeze-drying ability.

[0346] 1D Formulation - Results and Analysis Following testing of Formulations 1A-1C, a formulation window was identified that provided a favorable balance of isotonicity, solubility, and freeze-dryability. The formulations in Table 1D (Formulations 1D.1-1D.6), all of which had a pH of 5.5 and contained 10 mg / mL bottle-traffic alpha, were tested for freeze-dryability and osmolality in the same manner as Formulation 1C, with the results provided in Table 1I below. [Table 1I]

[0347] These results suggest that formulations with approximately 40 mM NaCl, or 20-50 mM NaCl, are optimal for the 10 mg / mL bottle-trough alfa formulation, because freeze-drying ability is slightly impaired at 60 mM NaCl, whereas at approximately 20 mM NaCl, bottle-trough alfa begins to precipitate, as observed upon dilution of samples for analytical purposes. Additionally, sugars such as trehalose (sucrose is also a reasonable alternative) at approximately 6% (w / v) (with a definite preference for ≥4.4% (w / v)) and exerting various modes of action (cryo / lyoprotectant, lyophilization bulking agent, and tonicity agent) are useful for formulating 10 mg / mL bottle-trough alfa as a lyophilized dosage form.

[0348] 1E Formulation - Results and Analysis Having established from the studies of Formulations 1A-1D that a formulation window providing a favorable balance of isotonicity and freeze-dryability, further studies were conducted to determine whether such a window also provided sufficient stability for the protein. To this end, the two formulations in Table 1E were subjected to short-term (3-week) stability studies (5°C, 25°C, and 40°C), which revealed that while both formulations (in both the lyophilized and liquid states) passed this stability study, the formulation with 75 mM NaCl and 4% trehalose at pH 5.5 did not freeze-dry sufficiently for the lyophilized formulation. The liquid formulation of the drug (10 mg / mL Vitrafus Alpha, 10 mM L-histidine, 5 mM methionine, 40 mM NaCl, 6% (w / v) trehalose, 0.05% (w / v) polysorbate 20, pH 5.5) did not demonstrate good stability (3 months at 5°C without significant changes in quality attributes; and at 6 months, there was an increase in subvisible particles, but other quality attributes remained well within expected ranges).

[0349] However, these initial stability studies suggested that liquid formulations of Bintrafus Alpha would not be stable for long periods of time, even when cooled to 5°C, and it was anticipated that a lyophilized formulation would provide a solution to this problem. However, the use of increased amounts of NaCl to solubilize Bintrafus Alpha at high concentrations has been found to have a detrimental effect on freeze-drying, and therefore many issues remain unresolved to enable formulations with relatively high drug concentrations.

[0350] That said, these studies demonstrate that when formulations present challenges in storage stability of certain liquid phases, lyophilized formulations offer a potential solution.

[0351] Conclusion of this Example Other formulation parameters such as buffer systems, surfactants, and other excipients can obviously be varied significantly without undesirably compromising the basic stability, solubility, freeze-dryability, and osmolality, provided that the pH and / or NaCl concentration are maintained within the appropriate window for a given concentration of Bottle Trough Alpha. For 10 mg / mL Bottle Trough Alpha, the following formulations have been found to be particularly suitable: [Table 1J]

[0352] Surprisingly, the best pH (pH 5.5) was within 1 pH unit of the pI of the TGFβR2 moiety, but a wider pH range, e.g., pH 5-9, appears to be tolerable, especially upon addition of sodium chloride.

[0353] Sodium chloride facilitated drug substance solubility, but too much NaCl can impair lyophilization ability.

[0354] However, at this stage, lyophilized formulations appear to offer the most potential for success. Indeed, lyophilized formulations offer a potential solution when formulations present certain liquid-phase storage stability challenges. However, if sodium chloride is selected as the ionic strength provider, lyophilized formulations may be limited in terms of maximum drug concentration, since higher drug concentrations require more NaCl to promote solubility and stability, but high salt concentrations somewhat impair lyophilization capabilities.

[0355] Example 2 - Relatively high concentration liquid formulation of IgG:TGFβR2 fusion protein General Overview While Example 1 provided a viable pharmaceutical composition, it was desirable to increase the concentration of the IgG:TGFβR2 fusion protein in both the lyophilized and liquid compositions and to determine whether these could withstand longer storage periods. To this end, further formulation studies were performed.

[0356] List of tested formulations Stability results indicated that the candidate formulation from Example 1 (2A.1) was also stable in liquid form, and therefore this formulation served as the reference sample in Example 2. To assess the feasibility of increasing the concentration of Bintrafus Alpha, a formulation screen was then performed on four additional formulations (2A.2-2A.5). These formulations are shown in Table 2A. [Table 2A]

[0357] Results and Analysis The formulations listed in Table 2A were subjected to long-term stability studies (up to 12 months at 5° C.), accelerated stability studies (up to 6 months at 25° C.), and stress stability studies (up to 3 months at 40° C.). Table 2B shows analytical results related to long-term stability studies (5° C.) with formulation 2A.2, which has a drug concentration of 20 mg / mL. [Table 2B]

[0358] Table 2C shows analytical results related to long-term stability studies (5° C.) with formulation 2A.3, having a drug concentration of 35 mg / mL. [Table 2C]

[0359] Table 2D shows analytical results related to long-term stability studies (5° C.) with formulation 2A.4, having a drug concentration of 40 mg / mL. [Table 2D]

[0360] Table 2E shows analytical results associated with long-term stability studies (5°C) and accelerated stability studies (25°C) with formulation 2A.5, which has a drug concentration of 40 mg / mL but an elevated sodium chloride concentration of 60 mM. [Table 2E]

[0361] Table 2F shows the protein stability by optical density for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5° C.), accelerated stability (25° C.), and stress stability (40° C.) at various time intervals ranging from time=0 to 12 months. No significant decrease in protein concentration was observed during these stability studies for any of the batches analyzed. [Table 2F]

[0362] Table 2G shows the % high molecular weight (%HMW) species by SE-UPLC for all four increasing concentration formulations (2A.2-2A.5) in Table 2A under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. With the exception of 2A.5 (60 mM NaCl), the higher concentrations, the higher %HMW, appeared more stable than the corresponding formulation with 40 mM NaCl (which also contained 40 mg / mL bottle trough alpha). This suggests that higher API concentrations require more NaCl to reduce aggregation. [Table 2G]

[0363] Table 2H shows the % low molecular weight (%LMW) species by CE-SDS for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. %LMW was lower for 2A.5 at all stability conditions, potentially indicating that NaCl may mitigate fragmentation in samples with higher drug concentrations. [Table 2H]

[0364] Table 2I shows the % oxidation levels by RP-UPLC for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. Similar % oxidation levels were observed across stability at 5°C ± 3°C and 25°C ± 2°C for all lots (considering the oxidation levels of the IRS reported for comparison in each session), but an increase was observed at 40°C ± 2°C. The high values ​​for 2-6 months at 5°C and 25°C may be misleading due to variability in analytical sessions. [Table 2I]

[0365] Table 2J shows the % deamidated form (LC) by IEX-HPLC for all four increasing strength formulations (2A.2-2A.5) in Table 2A under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. At 5°C ± 3°C and 25°C ± 2°C, all tested samples remained stable over time, increasing slightly only at the last time point for 2A.5. At 40°C ± 2°C, the %LC increased over time. The level of deamidation was comparable for all samples and apparently independent of the API concentration in the formulation. [Table 2J]

[0366] Table 2K shows the % purity by CE-SDS reduction for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. A slight decrease in purity was observed at 25°C ± 2°C, with a more significant decrease evident at 40°C ± 2°C. However, this trend was comparable across all formulations and appeared to be independent of concentration. [Table 2K]

[0367] Table 2L shows the % major clipping by CE-SDS reduction for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time = 0 to 12 months. The increase in % major clipping was similar for all formulations under all conditions, at all time points, and therefore appears to be independent of concentration. [Table 2L]

[0368] Table 2M shows the % anti-PD-L1 biological activity for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5°C), accelerated stability (25°C), and stress stability (40°C) at various time intervals ranging from time=0 to 12 months. No significant variation in the biological activity of the anti-PD-L1 moiety was observed over time under any stability condition. [Table 2M]

[0369] Table 2N shows the % TGF-β bioactivity for all four increasing strength formulations in Table 2A (2A.2-2A.5) under long-term stability (5° C.), accelerated stability (25° C.), and stress stability (40° C.) studies at various time intervals ranging from time=0 to 12 months. A significant decrease in the bioactivity of the TGF-β moiety was observed over time at 40° C.±2° C. to a similar extent for all formulations. [Table 2N]

[0370] conclusion Table 20 summarizes the trends observed throughout the stability studies described above. [Table 2O]

[0371] Again, it is expected that other formulation parameters such as buffer systems, surfactants, and other excipients may be varied significantly without undesirably compromising the basic stability, solubility, freeze-dryability, and osmolality, provided that the pH and / or NaCl concentration are maintained within the appropriate window for a given concentration of Bottle Trough Alpha. For 40 mg / mL Bottle Trough Alpha, the following formulations have been found to be particularly suitable: [Table 2P]

[0372] The major differences from the lead candidate identified in Example 1 are the higher concentration of vintrafus alpha, 40 mg / mL rather than 10 mg / mL, and the increased amount of sodium chloride from 40 mM to 60 mM. Furthermore, these studies suggested that the liquid formulations of Example 1 and this Example are indeed viable, and that lyophilization / reconstitution may not be strictly necessary, as storage of the liquid formulation is deemed more practical.

[0373] Further testing also showed that the optimal amount of NaCl for a given concentration of Bintrafus Alpha was as follows: 10-20mg / mL API can be stabilized by 40mM NaCl 40mg / mL, 60mM NaCl is generally preferred Trehalose at 60mg / mL is generally preferred, as is 100mM NaCl. 60mg / mL with or without trehalose, 150mM NaCl is generally preferred.

[0374] These studies also suggested that liquid formulations with high concentrations of IgG:TGFβR2 are indeed feasible, and that lyophilized formulations may even be preferable, especially when higher concentrations of sodium chloride are used.

[0375] Example 3A - Further Screening Tests - Step 1A (pH, Ionic Strength, and Protein Concentration Screens) General Overview Further formulation studies were conducted to evaluate variations in pH (pH 5.0-7.5), ionic strength (40-150 mM ionic salt, e.g., sodium chloride), and protein concentration (20, 40, and 60 mg / mL) in a 10 mM histidine buffer system, which served as an illustrative buffer system.

[0376] List of tested formulations An initial screen was performed on 20 formulations to establish feasible and optimal pH, feasible and optimal ionic strength, and feasible and optimal protein concentration. The formulations for this initial screen are shown in Table 3A. [Table 3A]

[0377] Results and Analysis The formulations in Table 3A were evaluated in a DoE study. Specifically, their unfolding temperature was tested. In addition, they were subjected to temperature stress (40°C for 4 weeks) and light stress (765 W / m2 The samples exposed to and stressed at 1000 K for 7 hours were analyzed by various analytical techniques.

[0378] For temperature-stressed samples, meaningful models were constructed with reference to the following analyses, although other additional analytical techniques were also used during preliminary analyses: HMW by SE-UPLC Isoform profiling by ICE3 Purity by CGE-SDS (reduced / non-reduced) / LMW Oxidized form by RP-UPLC Deamidated form by IEX-HPLC · Unfolding temperature by nano-DSC.

[0379] For light-stressed samples, meaningful models were constructed with reference to the following analyses, although other additional analytical techniques were also used during preliminary analyses: HMW by SE-UPLC Oxidized form by RP-UPLC Isoform profiling by iCE3.

[0380] Unfolding temperature Taken together, Figures 3 and 4 suggest that a pH range of 5.7–6.2 is optimal in terms of unfolding temperature, and that higher protein concentrations require higher ionic strength to provide conformational stability.

[0381] Table 3B shows the unfolding temperatures by nano-DSC for the formulations in Table 3A. [Table 3B]

[0382] temperature stress Table 3C shows the turbidity, as characterized by high values ​​of opacity (ranging from 7 NTU to 18 NTU), upon temperature stress testing of the formulations in Table 3A. No significant changes were observed for any of the samples after 4 weeks of incubation at +40°C ± 2°C. [Table 3C]

[0383] Table 3D shows the % deamidation during temperature stress testing of the formulations in Table 3A. At TO, no significant differences were observed for any sample compared to the standard, except for samples with high pH and relatively low ionic strength relative to the API concentration—e.g., sample #15, which has a higher % deamidation value than the others, likely due to the synergistic effect of high protein concentration, high pH, ​​and low ionic strength. On the other hand, after 4 weeks of temperature stress at +40°C ± 2°C, increasing pH correlates with increasing % deamidation. Overall, ionic strength and protein concentration had little effect on % deamidation. A pH range of 5.0–6.2 appears to be optimal, while formulations with a pH between 7.1–7.5 perform less well in this setting. [Table 3D]

[0384] Figures 5 and 6 together suggest that ionic strength and protein concentration have little effect on %LMW species after temperature stress, whereas pH has a significant effect, with the pH region of 5.3-6.7 clearly optimal in this situation. However, lower ionic strength correlates with fragmentation at similar pH levels.

[0385] Figures 7 and 8 together suggest that the ionic strength should be increased as the protein concentration increases to avoid aggregate formation and ultimately protein precipitation. Furthermore, a pH between 5.0 and 6.6 is clearly optimal in terms of % HMW species after temperature stress.

[0386] In all species monitored by CGE (under reducing conditions), there was no significant difference in purity between samples at time = 0, except that sample #19 increased slightly in major clipping.

[0387] At 40 °C during 4 weeks of temperature stress, all samples with pH > 7 or pH < 5.5 showed lower overall purity, independent of ionic strength and protein concentration. Low pH (pH 5.0 and to a lesser extent also pH 5.3) caused an increase in impurities at peak 7, and also an increase in major clipping (peak 6) was observed under low pH conditions (<pH 5.5).

[0388] At time = 0, cIEF showed no significant difference between samples.

[0389] Figure 9 and data elsewhere suggest that the effects of ionic strength and protein concentration on isoforms are not significant after temperature stress, while the isoform distribution is affected by pH. Data from Figure 9 suggest that pH 5.3 - 5.9 is optimal in terms of the isoform situation.

[0390] Table 3E shows the isoform clusters after 4 weeks of temperature stress at 40 °C. <​​​​​​​​​​​​​Table 3F shows the %HMW species before and after light stress. Light stress increases %HMW for all samples, but there appears to be a slightly lower level of aggregation between pH 5.2 and 6.6, and a significant effect of ionic strength. [Table 3F]

[0393] The cIEF measurements of the samples exposed to light stress suggest that, with the exception of samples 8, 15, 16, 18, and 19, they show no significant differences compared to the reference standard.

[0394] Table 3G shows the isoform clusters after light stress. [Table 3G]

[0395] Figures 12, 13 and 14 together suggest that neither ionic strength, protein concentration, nor even pH significantly affect % oxidation under light stress conditions.

[0396] conclusion Figure 15 shows 2D and 3D contour plots of desirability parameters (reflecting the balance of factors in overall response assessment) expressed as contour lines (in 2D plots) and surfaces (in 3D plots) within the formulation space (with a fixed protein concentration of 20 mg / mL) at various pHs and ionic strengths (given as mM NaCl). This suggests that the optimum conditions for IgG:TGFβR2 at this concentration are pH 5.4-6.1, such as pH 5.7, and ionic strength 40-60 mM NaCl, such as 40 mM NaCl. Thus, for a pharmaceutical composition containing 10-30 mg / mL IgG:TGFβR2 fusion protein, a pH of 5.2-6.3 is desirable, along with a minimum ionic strength, such as 10 mM NaCl, and preferably an ionic strength range of 10-80 mM NaCl.

[0397] Figure 16 shows 2D and 3D contour plots of desirability parameters (reflecting the balance of factors in overall response assessment) expressed as contour lines (in 2D plots) and surfaces (in 3D plots) within the formulation space (with a fixed protein concentration of 40 mg / mL) at various pHs and ionic strengths (given as mM NaCl). This suggests that the optimum conditions for IgG:TGFβR2 at this concentration are pH 5.7-6.1, such as pH 5.9, and ionic strengths of 50-70 mM NaCl, such as 60 mM NaCl. Thus, for a pharmaceutical composition containing 30-50 mg / mL of IgG:TGFβR2 fusion protein, a pH of 5.5-6.5 is desirable, along with a minimum ionic strength of, for example, 20 mM NaCl, and preferably an ionic strength range of 30-90 mM NaCl.

[0398] Figure 17 shows 2D and 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) expressed as a contour line (in the 2D plot) and a surface (in the 3D plot) within the formulation space (with a fixed protein concentration of 60 mg / mL) at various pHs and ionic strengths (given as mM NaCl). This suggests that the optimum for IgG:TGFβR2 at this concentration is pH 5.7-6.1, such as pH 5.9, and an ionic strength of 130-150 mM NaCl, such as 150 mM NaCl. It has also been found that the optimum for IgG:TGFβR2 at this concentration is pH 5.7-6.1, such as pH 5.9, and an ionic strength of 80-90 mM NaCl, such as 80 mM NaCl. Thus, for a pharmaceutical composition containing 50-70 mg / mL of IgG:TGFβR2 fusion protein, a pH of 5.5-6.5 is desirable, along with a minimum ionic strength, for example 50 mM NaCl, and preferably an ionic strength in the range of 60-200 mM NaCl.

[0399] Example 3B - Further Screening Tests - Step 1B (Buffer Screen) General Overview Following the results of Example 3A, experiments were conducted to both confirm the results of Example 3A and explore alternative buffers. Thus, three buffer types were selected, covering a pH range of 5.7 to 6.2. Ionic strength was optimized based on protein concentration according to the results of Example 3A.

[0400] List of tested formulations Table 3H shows the 12 formulations and the reference formulation tested in this particular screen. [Table 3H]

[0401] Results and Analysis The formulations listed in Table 3H were subjected to temperature stress conditions (40°C for 4 weeks) and light stress conditions (765 W / m 2 Temperature-stressed samples were tested by the following methods, but only the methods in bold revealed significant differences: [Table 3H2]

[0402] At the same time, the light-stressed samples were tested as follows: HMW by SE-UPLC Isoform profiling by ICE3 Oxidized form by RP-UPLC · Purity / LMW by CGE-SDS (reduced / non-reduced).

[0403] The qualitative results for both temperature and light stress conditions combined are shown in Table 3I below.

[0404] Table 3I shows the qualitative results (pass or fail) of both the temperature and light stress tests for all formulations in Table 3H. [Table 3I]

[0405] conclusion These results suggest that histidine is the most stabilizing of the three tested buffers containing high protein concentrations, but that other buffers, especially succinate, are highly suitable under suitable conditions. Furthermore, these studies highlight the feasibility of operating at relatively high protein concentrations, such as 40 and 60 mg / ml.

[0406] Example 3C - Further Screening Studies - Step 2A (Excipient Screening) General Overview Following the results of Example 3B, an excipient screen was performed, keeping the buffer fixed at 10 mM histidine, pH 5.9, while varying the ionic strength from 60 mM to 100 mM and the protein (Vintrafus alpha) concentration from 40 to 60 mg / mL. 5 mM methionine was also included in all formulations in this example to mitigate the oxidative effects evident from the previous analysis.

[0407] The excipients screened were: Sugar polyols, such as mannitol, trehalose, sorbitol; amino acids, such as arginine, lysine, proline, glutamic acid; and Surfactants such as Tween 20 and Corifol 188.

[0408] Before being subjected to further analysis, the relevant formulations were stress tested in the following manner: Temperature stress (up to 12 weeks at 40°C) Light stress (765W / m 2 (7 hours later) F / T stress (after three cycles from -25°C to RT) Mechanical stress (after 3 days at 300 rpm).

[0409] List of tested formulations Table 3J shows the 48 formulations tested in this particular screen. [Table 3J-1] [Table 3J-2]

[0410] Results and Analysis The formulations listed in Table 3J were tested under temperature stress conditions (40°C for 4 and 8 weeks), light stress conditions (765 W / m 2 The cells were examined under freeze-thaw stress (three F / T cycles from -25°C to room temperature) and mechanical stress (300 rpm for 3 days).

[0411] The temperature stressed samples were tested by the following method: [Table 3J2]

[0412] The light-stressed samples were tested by the following method: [Table 3J3]

[0413] The F / T and mechanically stressed samples were tested by the following methods: HMW by SE-UPLC · Purity / LMW by CGE-SDS (reduced / non-reduced).

[0414] temperature stress Figures 18 and 19 together suggest that protein concentration has a significant effect on % HMW under temperature stress, but 50 mM arginine and 100 mM lysine are the best performing "swing excipients" over 4 weeks, while 100 mM arginine and 100 mM lysine perform best over 8 weeks of temperature stress.

[0415] Figures 20 and 21 together suggest that sorbitol (at 40 and 50 mg / mL protein concentrations) and mannitol (at 60 mg / mL protein concentration) show the highest primary clipping, 100 mM arginine shows the highest %LMW, and trehalose shows the lowest, and that protein concentration has no significant effect after 4 weeks under temperature stress.

[0416] FIG. 22 suggests that trehalose and 50 mM arginine show the lowest % deamination under temperature stress, while L-glutamic acid, lysine and proline show the highest % deamination.

[0417] 23 and 24 together suggest that polysorbate 20 performs better than corifol 188, and that sorbitol actually reveals the lowest % oxidized forms after 4 weeks of temperature stress, whereas 50 mM L-arginine is evident showing the highest oxidized forms at 8 weeks of temperature stress (protein concentration was fixed as it was shown to have no effect).

[0418] Figure 25 suggests that proline was closest to the target % Cluster 1 during temperature stress, which was 52% (protein concentration was fixed as it was shown to have no effect).

[0419] FIG. 26 suggests that mannitol and lysine exert a lower % Cluster 2 than the target, which is 12%, upon temperature stress.

[0420] Light stress FIG. 27 suggests that under light-stressed conditions, 100 mM arginine and lysine exerted the lowest %HMW, and that protein concentration significantly affected %HMW.

[0421] Figures 28 and 29 together suggest that under light stress conditions, 100 mM arginine, sorbitol, and mannitol (the latter two at only 60 mg / mL protein concentration) exerted the highest % primary clipping, while 50 mM arginine exerted the highest % LMW.

[0422] Figure 30 suggests that 100 mM arginine provides the lowest % oxidation under light stress conditions.

[0423] Freeze-thaw stress FIG. 31 suggests that under freeze-thaw stress, mannitol exerts the highest %HMW, and protein concentration does not significantly affect %HMW.

[0424] Mechanical stress Figures 32 and 33 together suggest that polysorbate 20 is superior to corifol 188 in preventing aggregation under mechanical stress (especially in combination with sorbitol, arginine, and lysine).

[0425] General Desirability The overall desirability of the various excipients was calculated based on a balance of factors, stress tests, and results.

[0426] Figure 34 suggests that formulations with protein concentrations of 40 or 50 mg / mL have a higher overall desirability compared to 60 mg / mL.

[0427] For a formulation having a 40 mg / mL protein concentration, a particularly desirable polyol is trehalose, and particularly desirable amino acids are 50 mM arginine and lysine.

[0428] For a formulation having a 50 mg / mL protein concentration, a particularly desirable polyol is trehalose, and particularly desirable amino acids are 50 mM arginine and lysine.

[0429] For a formulation having a 60 mg / mL protein concentration, a particularly desirable polyol is trehalose, and particularly desirable amino acids are 50 mM arginine and lysine.

[0430] Figure 35 suggests a slightly higher desirability of polysorbate 20 compared to corifol 188 across virtually all conditions.

[0431] Example 3D - Step 2B (Excipient Combinations and Fine Tuning) General Overview Based on the overall desirability results of step 2A, it was decided to continue with protein concentrations of 40 and 50 mg / mL in preparing the trehalose and arginine / lysine combination formulations and to explore the synergistic effect between sugars and amino acids.

[0432] List of tested formulations Table 3K shows the 12 formulations (+ reference) tested in this particular screen. [Table 3K]

[0433] Results and Analysis The formulations listed in Table 3K were tested under temperature stress conditions (40°C for 4, 8, and 12 weeks), light stress conditions (765 W / m 2 The samples were examined under mechanical stress (300 rpm for 3 days) as well as under high-temperature conditions (7 h at 300 rpm).

[0434] The temperature stressed samples were tested by the following method: [Table 3K2]

[0435] The light-stressed samples were tested by the following method: [Table 3K3]

[0436] The mechanically stressed samples were tested according to the following method: HMW by SE-UPLC · Purity / LMW by CGE-SDS (reduced / non-reduced).

[0437] Table 3L shows the nano-DSC results for the 12 formulations (+ reference) tested in this particular screen. [Table 3L]

[0438] Table 3L shows that formulations #89 and #91 have relatively high T m The increased Tween 20 concentration appears to have a stabilizing effect compared to #89 and #90, suggesting a higher conformational stability of these formulations.

[0439] Figure 36 suggests that: the increase in %HMW is comparable for the L-arginine- and L-lysine-containing combinations; slightly higher values ​​are observed for the 50 mg / ml protein concentration formulations with excipient combinations (#96, #97); even higher values ​​are obtained for formulations with only one excipient (#100 and #101), and slightly greater values ​​for the formulation containing EDTA. The results for formulations #89-#95 are better than those for 2A.5 in Table 2A (same protein concentration).

[0440] Figure 37 suggests that the increase in %LMW was similar for nearly all formulations, but was slightly higher for the EDTA-containing formulation.

[0441] Figure 38 suggests that after 13 weeks of temperature stress there were no significant differences in oxidation, with only slightly lower values ​​in the EDTA-containing formulation.

[0442] Figure 39 suggests that all formulations exhibited similar deamidation after temperature stress.

[0443] Figure 40 suggests that purity decreased slightly across all formulations, although comparable to each other.

[0444] Figure 41 shows that after temperature stress, the main clipping (peak 6) increased in all formulations, with the highest increase observed for the EDTA-containing formulation (#98) and for the trehalose-only formulations with lower NaCl levels (#99 and reference). These same formulations were also characterized by higher values ​​of peak 7 contaminants.

[0445] Figure 42 indicates that at the higher protein concentration of 50 mg / ml, a relatively high increase in %HMW is observed after light stress for the lysine-containing; EDTA-containing; and single excipient-containing formulations.

[0446] Figure 43 indicates that % oxidation increased after light stress for all formulations, with slightly higher values ​​observed for the EDTA-containing formulation and slightly lower values ​​observed for the trehalose-only formulation (#101).

[0447] Figure 44 suggests that for all formulations, LMW levels are not significantly different after light stress.

[0448] Figure 45 suggests that there was no significant change in purity after light stress, except that formulation #99 showed a slightly greater decrease after stress.

[0449] Figure 46 suggests that there is no significant change in the level of % major clipping after light stress.

[0450] Figure 47 suggests that there was only slight variation in %HMW after mechanical stress, but the starting values ​​were relatively high for the EDTA-containing formulations, and more significant increases were observed in the case of formulations #91 and #95.

[0451] Figure 48 suggests that there was no significant change in %LMW after mechanical stress, but only formulations #89 and #94 showed a moderate increase in LMW.

[0452] Figure 49 suggests that for formulation #95, the % purity decreased significantly after mechanical stress, while only minor variations were observed for the other formulations.

[0453] Figure 50 suggests that there was no significant variation in % primary clipping after mechanical stress, except for formulation #95, which contained the highest amount of lysine.

[0454] conclusion From the data collected in this formulation screen, the best performing formulations under all conditions were those with excipient combinations, particularly trehalose in combination with L-arginine. Two formulations with a protein concentration of 50 mg / ml performed comparable to the 40 mg / ml one, indicating that higher concentrations are indeed feasible. 2A.5 in Table 2A, containing 40 mg / ml Bottle Lavs Alpha and 60 mM NaCl, was used as a comparison in this temperature stress study and appeared to be largely equivalent to the formulations with the excipient combinations.

[0455] Overall, 2A.5 in Table 2A, formulation #89, formulation #91, and formulation #96 were considered to be particularly promising candidate formulations.

[0456] Example 4 - Further formulation testing General Overview The overall plan for this experiment was to initially screen samples at 20-25 mg / mL bottle trough alpha concentrations to evaluate pH, buffer, and excipients. Then, concentration escalation and short-term stability studies were performed to select formulations, followed by surfactant screening and short-term stability studies of 4-5 formulations at a final higher concentration.

[0457] List of tested formulations Table 4A shows the 20 formulations (F1-F20), including the reference formulation (F1), to be tested in this particular screen. [Table 4A]

[0458] Results and Analysis The formulations were stored and tested according to the following outline shown in Table 4B (tests performed are marked with an "x"). [Table 4B]

[0459] In this context, TO is time 0; T-FT is after three freeze-thaw cycles (Figure 51); T-2w and T-4w mean after 2 weeks and 4 weeks, respectively, at either 25°C or 40°C.

[0460] FIG. 52 is a bar graph showing how pH varies at various time points during stress testing for all of formulations F1-F20, including (bars are listed from left to right for each formulation): time=0; after 3FT 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.

[0461] The target pH for each formulation is shown in Table 4A, and Figure 52 illustrates how this varies.

[0462] For all formulations, the target pH value (±0.1) was reached at TO.

[0463] After 4 weeks of storage, the pH value remained unchanged in most of the formulations.

[0464] Lower pH values ​​were detected in the three formulations at a protein concentration of 50 mg / ml: F12 (His buffer, pH 5.7), stored at 40°C for 4 weeks F17 (buffer-free, pH 5.7), stored at 40°C for 4 weeks F19 (containing sorbitol, pH 6.0) stored at 25°C for 2 and 4 weeks.

[0465] FIG. 53 is a bar graph showing the osmolality for each of formulations F1-F20.

[0466] All formulations reached osmolality values ​​within the specified range of 260-340 mOsmol / kg (green line) - except for F15 (where a lower osmolality value of 254 mOsmol / kg was detected).

[0467] Figure 54 is a bar graph showing how turbidity varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0468] Turbidity appeared to depend on protein concentration - higher turbidity values ​​were detected in samples with higher protein concentrations. Turbidity values ​​generally remained unchanged for most formulations, but increases of 0.5 to 2.0 were observed in the following formulations: T4w-25°C, F3 (His buffer, pH 5.7) F13 (His buffer, pH 6.0) at T2w-25°C and T2w-40°C F17 (buffer-free, pH 5.7) at T2w-25°C and T4w-25°C.

[0469] Increases of 4.1 to 7.4 were observed for the following formulations: F19 (containing sorbitol, pH 6.0) at T2w-25°C and T4w-25°C.

[0470] Visual inspection was performed and reported according to the codes shown in Tables 4C and 4D. [Table 4C] [Table 4D]

[0471] The results of the visual inspection under the various aforementioned stress conditions and time points are shown below in Tables 4E, 4F, and 4G. [Table 4E] [Table 4F] [Table 4G]

[0472] The results of the analysis performed according to Table 4A are shown in Table 4H. [Table 4H-1] [Table 4H-2] [Table 4H-3] [Table 4H-4]

[0473] The analytical results in Table 4H were classified based on the thresholds defined in Table 4J (see superscript numbers in Table 4H). [Table 4I]

[0474] Regarding UV spectrophotometric (SoloVPE) analysis to determine bin trough alpha concentrations, it is noted that all target concentrations were reached at time = 0. No significant changes were observed after stress application.

[0475] Relatively low viscosities of approximately 1.0-1.7 mPa*s were detected for all formulations, and relatively high viscosity values ​​(1.6-1.7 mPa*s) were observed in formulations containing a protein concentration of 50 mg / ml (F9-F20).

[0476] Minimal subvisible particles were detected in all formulations at TO. After 2 and 4 weeks, an increase in subvisible particles was observed for F17 (buffer-free system, pH 5.7) and F19 (sorbitol-containing formulations) stored at 25°C, and for F8 (sorbitol-containing formulation) stored at 40°C. After 4 weeks of storage at 25°C and 40°C, F5 showed the greatest increase in subvisible particles. For most of these formulations, low particle concentrations were detected (<1,000 particles ≥ 5 μm).

[0477] Regarding the HP-SEC results, an LMW shoulder was noted to develop for F19 after 4 weeks of storage at 25°C. However, LMW, unlike HMW, was not integrated into the overall monomer content results. In general, a decrease in monomer content and an increase in HMW content were observed for all formulations after up to 4 weeks of storage at 25°C and 40°C. Arginine-containing formulations showed a smaller decrease in monomer content compared to other formulations at higher protein concentrations. At higher protein concentrations, pH 6.0 appears to be superior to pH 5.5 and 5.7 (tested with His as the buffer system). Comparison of F16 and F20 suggests that more arginine and less NaCl have a stabilizing effect on the protein.

[0478] Regarding deamidation (assessed by IEX-HPLC, % Peak 2 content - Peak 2 is the largest peak and any decrease in the size of Peak 2 indicates deamidation), a relative peak area of ​​>93% was measured for Peak 2 at TO for all formulations. A decrease in the relative peak area of ​​Peak 2 was observed for all formulations after storage, which was more pronounced at 40°C than at 25°C - thus an increase in the peak area of ​​the "LC Deam Peak" group (which is the merger of all peaks except Peak 2) was observed.

[0479] A slight increase in the relative peak area of ​​the "LC Deam peak" was also observed after FT, which was equivalent to 2 weeks of storage at 25° C. For the higher concentration formulations, the most promising results were obtained for: 25mg / ml protein concentration: His buffer system, pH 5.5 and 5.7 (F2 and F3) Arginine and sorbitol-containing preparations (F7 and F8) • 50mg / ml protein concentration: His buffer system, pH 5.5 and 5.7 (F11 and F12).

[0480] Figure 55 is a bar graph showing how the temperature of the Tm2 peak of the nano-DSC traces varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0481] Figure 56 is a bar graph showing how the onset temperature, Tm, of the nano-DSC traces varies at various time points during stress testing for all of formulations F1-F20, including (bars are 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.

[0482] Figures 55 and 56 together suggest that Tm2 values ​​above 66°C were detected for all formulations and did not change significantly upon stress or storage.

[0483] There were no significant changes in the isoform profile. At time=0, the following relative peak areas were detected: -> Cluster 1: approx. 56% -> Cluster 2: approx. 11% -> Cluster 3: approx. 21% ->Cluster 4: Approximately 12%.

[0484] Freeze-thaw does not appear to affect the isoform profile of these samples. After 4 weeks of storage at 40°C, the following was noted: -> Decrease in relative peak area of ​​cluster 2, ->Increase in the relative peak area of ​​cluster 4.

[0485] cGE was performed under both reducing and non-reducing conditions, and the results are tabulated in Table 4H. Under reducing conditions, very similar results were obtained for all formulations at time = 0 (a difference of 0.6% between maximum and minimum purity [%]). The purity of all formulations decreased after 4 weeks of storage at 40°C, but such decrease was least significant for F16, F18, and F20, and most significant for F1, F8, F12, and F17. The decrease in purity was mainly achieved by increased primary clipping [%]. In addition, the content of other contaminants increased, but these had little impact on the overall purity of the formulations.

[0486] For cGE under non-reducing conditions, very similar results were observed for all formulations at time = 0 (a 1.1% difference between the maximum and minimum corrected area [%] of the intact molecule). The relative peak area of ​​the intact molecule for all formulations decreased after 4 weeks of storage at 40°C, but such a decrease was least pronounced for F5, F6, F14, and F15, and most pronounced for F2, F19, and F12. The decrease in the corrected peak area of ​​the intact molecule was accompanied by an increase in the relative peak area of ​​the LMW species (T4w_40°C).

[0487] Oxidation measured by RP-UPLC is reported in Table 4H. A concomitant decrease in intact Met516 content with an increase in oxidized Met516 content was observed for most samples after 4 weeks of storage at 40°C, but was most pronounced for F4, F9, and F15.

[0488] conclusion At time = 0, the target pH, concentration, and osmolality values ​​were reached for all formulations F1-F20. A small number of visible and subvisible particles were observed, and viscosity was relatively low for all formulations. By HP-SEC, the main peak accounted for 98% in all formulations. By cGE, purity was approximately 94% for all formulations under reducing conditions and greater than 96% for all formulations under non-reducing conditions.

[0489] Freeze-thaw (FT) stress did not significantly affect the stability of these formulations. Furthermore, pH, concentration, number of visible and subvisible particles, turbidity, and monomer content remained substantially unchanged after FT stress.

[0490] Samples stored at 25°C and 40°C for up to 4 weeks exhibited the following characteristics: pH did not change substantially Turbidity values ​​increased, with higher values ​​observed for samples stored at 25°C compared to samples stored at 40°C. More visible particles were observed after 4 weeks of storage at 25°C and 40°C Protein concentrations remained unchanged A relatively high number of invisible particles was observed for F5, F8, F17 and F19. Arginine-containing formulations showed a smaller decrease in monomer content over time (HP-SEC) · cGE: Purity (%, reduced conditions) as well as content of intact molecules (%, non-reduced conditions) of all formulations decreased after 4 weeks of storage at 40°C -> F12 showed the worst performance for both parameters.

[0491] Finally, % oxidation measured by RP-UPLC showed that intact Met516 was 96-97% for all formulations at time = 0, and that a decrease in intact Met516 and an increase in oxidized Met516 was observed after 4 weeks of storage at 40°C.

[0492] Formulations containing 25 mg / mL Bintrafus Alpha (F2-F8) appeared to perform best overall at pH 5.5, while citrate buffer was noted to perform best at pH 6.0. A beneficial effect was also observed at this concentration when arginine was included in the formulations.

[0493] Formulations containing 50 mg / mL Vitrafus Alpha (F9-F20) appeared to perform best overall at pH 5.5, but at this concentration, histidine buffer appeared to perform best at pH 6.0 compared to the other buffers. Arginine was also noted to be beneficial at lower concentrations.

[0494] Further testing is currently underway on the following promising candidates: F13, F16, F20, and F21-F23, as detailed in Table 4K. [Table 4K]

[0495] Example 5 - Further formulation testing General Overview To further check the suitability of the ionic tonicity agent in combination with the sugar and / or amino acid moieties to stabilize the IgG:TGFβR fusion protein, various IgG:TGFβR fusion proteins were tested in such formulations, particularly IgG:TGFβR fusion proteins with different IgG classes, different paratopes, and different TGFβR moieties.

[0496] List of IgG:TGFβR fusion proteins tested The following three anti-PD-L1 (IgG):TGFβR2 fusion proteins were tested: Vintrafs alpha (light and heavy chain sequences correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively) Fusion protein 2 (light and heavy chain sequences correspond to SEQ ID NO: 33 and SEQ ID NO: 34, respectively) Fusion protein 3 (light and heavy chain sequences correspond to SEQ ID NO: 15 and SEQ ID NO: 18, respectively).

[0497] List of tested formulations 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): [Table 5A]

[0498] Results and Analysis The formulations listed in Table 5A were subjected to stressed (40° C. for 4 weeks) stability studies.

[0499] Figure 57 is a bar graph showing % high molecular weight (% HMW) species by SE-UPLC. Consistent with previous results and further confirmed by statistical tabulation, arginine reduces aggregation, and increasing amounts of NaCl similarly stabilize the protein (even in the absence of arginine).

[0500] Figure 58 is a bar graph showing % low molecular weight (%LMW) species by CGE-NRED. Consistent with previous results and further confirmed by statistical tabulation, trehalose prevents fragmentation and arginine tends to increase it, although the effect of arginine may be masked by the stabilizing effect of trehalose.

[0501] Table 5B shows the unfolding temperatures by micro-DSC. No significant changes were observed in the unfolding temperatures for any of the formulations tested. [Table 5B]

[0502] The three-dimensional structure of these samples was determined by fluorescence, and no loss of three-dimensional structure was observed in any of the samples.

[0503] conclusion The collected data was further analyzed for each fusion protein by desirability plots.

[0504] Figure 59, for example, shows 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) represented as a surface in formulation space with varying trehalose concentration and ionic strength (given as mM NaCl) for each of the anti-PD-L1(IgG):TGFβR2 fusion proteins tested. Arginine was maintained at a constant concentration of 100 mM in these plots, as this was found to be the most desirable (see also Figures 60 and 61).

[0505] Figure 60 shows 3D contour plots of the desirability parameter (reflecting the balance of factors in the overall response assessment) expressed as a surface within a formulation with varying arginine concentration and ionic strength (given as mM NaCl) for each of the anti-PD-L1(IgG):TGFβR2 fusion proteins tested. Trehalose was maintained at a constant concentration of 200 mM in these plots, as this was found to be the most desirable (see also Figures 59 and 61).

[0506] Figure 61 shows 3D contour plots of the desirability parameters (reflecting the balance of factors in the overall response assessment) expressed as a surface within a formulation with varying concentrations of arginine and trehalose for each of the anti-PD-L1(IgG):TGFβR2 fusion proteins tested. Ionic strength (given as mM NaCl) was maintained at a constant concentration of 100 mM in these plots, as this was found to be most desirable (see also Figures 59 and 60).

[0507] Overall, it was found that it was desirable to use increasing amounts of ionic strength, amino acid moieties, and disaccharides for the anti-PD-L1(IgG):TGFβR2 fusion proteins tested, as also reflected by the desirability plots, demonstrating the desirability of using increasing concentrations of all three components.

[0508] General summary of conclusions Example 1 suggests the following: A pH relatively close to the pI of one of the key protein domains (i.e., the TGFβR2 portion) surprisingly has advantages for the solubility and stability of the protein. A minimum ionic strength (preferably provided by sodium chloride) is preferred to promote protein solubility. Higher ionic strength is generally required to support higher protein concentrations, at least from the standpoint of protein solubility. Lyophilization facilitates long-term storage of the formulation, and the lyophilized formulation can be easily reconstituted. Trehalose facilitates lyophilization, but alternatives may be considered. If too much sodium chloride is used to achieve higher ionic strength, e.g., to support higher protein concentrations, lyophilization becomes more challenging. In such cases, alternative ionic strength providers may be considered.

[0509] Example 2 suggests the following: The formulation of Example 1 is indeed viable in terms of long-term storage stability when formulated in liquid form. Protein concentrations can be increased while maintaining viability for long-term storage stability, especially in liquid formulations. Formulations with higher concentrations of relevant proteins tend to benefit from higher ionic strength (e.g., higher NaCl concentrations). Surprisingly, NaCl also aids in protein solubility and protein stabilization. Histidine is apparently the preferred buffer for these formulations, but alternatives may be considered.

[0510] Example 3 suggests the following: Higher protein concentrations generally require the support of higher ionic strength, which reinforces previous findings. Higher protein concentrations benefit from a slightly higher pH, which is still closer to the pI of the TGFβR2 group than would normally be expected. A variety of buffers are viable as long as the pH is appropriate for the protein and buffer, but the best pH will vary slightly for different buffers. Histidine buffers appear to provide the best performance. With regard to various stress conditions (temperature, light, mechanical, freeze-thaw), some excipients perform better than others, and this can vary with various factors such as fragmentation, conformational stability, aggregation, isoforms, deamidation, oxidation, turbidity, etc. Although arginine, trehalose, and lysine appear to be the best performing excipients, their relative ranking to each other varies with protein concentration. Trehalose is generally superior to other sugars such as mannitol and sorbitol. Arginine has proven to be an excellent stabilizer, especially against aggregation. Polysorbate 20 appears to be slightly superior to Corifol 188 surfactant, especially in terms of reducing aggregation. Overall, combinations of trehalose and arginine at various relative molar ratios proved advantageous.

[0511] Example 4 suggests the following: · Arginine can potentially (partially) replace NaCl as a source of ionic strength. · Buffer-free systems appear less favorable. A slightly higher pH appears to work better for higher protein concentrations.

[0512] Example 5 further confirms that increasing amounts of ionic strength, amino acid moieties, and sugars are beneficial for stabilization of IgG:TGFβR fusion proteins.

Claims

1. 1. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an ionic tonicity agent that is sodium chloride, a disaccharide that is trehalose, a buffer system that is a histidine buffer system, and a non-ionic surfactant that is polysorbate 20; the light chain and heavy chain sequences 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; And the pharmaceutical composition is a. characterized by a pH of 5.3-5.7 and containing 5-15 mg / mL of said IgG:TGFβR fusion protein, 5-15 mM of said buffer system, 30-50 mM of said ionic tonicity agent; 150-170 mM of said disaccharide, and 0.3-0.7 mg / mL of said non-ionic surfactant; or b. A pharmaceutical composition characterized by a pH of 5.3-5.7 and comprising 35-45 mg / mL of said IgG:TGFβR fusion protein, 5-15 mM of said buffer system, 50-70 mM of said ionic tonicity agent; 150-170 mM of said disaccharide, and 0.3-0.7 mg / mL of said non-ionic surfactant.

2. A pharmaceutical composition comprising an IgG:TGFβR fusion protein, an ionic tonicity agent which is sodium chloride, a disaccharide which is trehalose, a buffer system which is a histidine buffer system, a non-ionic surfactant which is polysorbate 20, an amino acid component which is arginine, and an antioxidant which is methionine, the light chain and heavy chain sequences 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; And the pharmaceutical composition is c. characterized by a pH of 5.7-6.1 and containing 35-45 mg / mL of the IgG:TGFβR fusion protein, 5-15 mM of the buffer system, 50-70 mM of the ionic tonicity agent; 90-110 mM of the disaccharide, 40-60 mM of the amino acid component, and 0.3-0.7 mg / mL of the non-ionic surfactant; d. characterized by a pH of 5.7-6.1 and containing 35-45 mg / mL of the IgG:TGFβR fusion protein, 5-15 mM of the buffer system, 50-70 mM of the ionic tonicity agent; 65-85 mM of the disaccharide, 65-85 mM of the amino acid component, and 0.3-0.7 mg / mL of the non-ionic surfactant; e. characterized by a pH of 5.7-6.1 and containing 45-55 mg / mL of the IgG:TGFβR fusion protein, 5-15 mM of the buffer system, 90-110 mM of the ionic tonicity agent, 40-60 mM of the disaccharide, 40-60 mM of the amino acid component, and 0.3-0.7 mg / mL of the non-ionic surfactant; f. characterized by pH 5.5 and containing 10 mg / ml of Vintrafus alpha, 10 mM histidine, 40 mM sodium chloride, 159 mM trehalose, 0.5 mg / ml of polysorbate 20, 5 mM methionine; g. characterized by a pH of 5.5 and containing 40 mg / ml of Vitrafus alpha, 10 mM histidine, 60 mM sodium chloride, 159 mM trehalose, 0.5 mg / ml of polysorbate 20, 5 mM methionine; h. characterized by a pH of 5.9 and containing 40 mg / ml of Vitrafus alpha, 10 mM histidine, 60 mM sodium chloride, 100 mM trehalose, 50 mM arginine, 0.5 mg / ml of polysorbate 20, and 5 mM methionine; or i. A pharmaceutical composition characterized by a pH of 5.9 and containing 50 mg / ml of Vintrafus alpha, 10 mM histidine, 100 mM sodium chloride, 50 mM trehalose, 50 mM arginine, 0.5 mg / ml of polysorbate 20, 5 mM methionine.

3. The pharmaceutical composition according to claim 1 or 2, further comprising a surfactant.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the IgG:TGFβR fusion protein has the amino acid sequence of vintrafs alpha.

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