Aqueous Compositions of Engineered Protein Constructs Containing an Fc Domain

Abstract

In particular, an aqueous solution composition having a pH in the range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality regulator Includes; The aqueous solution composition is provided, wherein the buffer is present in the composition at a total concentration in the range of about 0 mM to about 10 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM.

Description

[Technical field]

[0001] The present invention relates to aqueous solutions of engineered protein constructs containing an Fc domain, with low buffer concentration and low ionic strength. [Background technology]

[0002] (background) Engineered protein constructs containing Fc domains are widely used in therapy. The Fc domain is the C-terminal region of an antibody that interacts with cell surface receptors called Fc receptors and with some proteins of the complement system, thereby activating the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein chain fragments, each derived from the second and third constant domains of the antibody heavy chain. In IgM and IgE antibody isotypes, the Fc domain is composed of two identical protein chain fragments, each derived from the second, third, and fourth constant domains of the antibody heavy chain. The molecular weight of the Fc domain can usually be in the range of 25-40 kDa, and can be larger if glycosylation is present. A wide range of physiological effects result from immune system activation mediated by antibody Fc domain binding, including cytolysis and degranulation of mast cells, basophils, and eosinophils.

[0003] Various engineered antibody protein constructs, such as bispecific and trispecific antibodies, have been developed, including some engineered protein constructs in which the Fc, separated from the Fab portion of the antibody molecule (the portion that confers antigen-binding specificity), can serve a purpose different from its physiological purpose, in particular to extend the in vivo half-life of the protein construct.

[0004] When formulated as aqueous solutions, proteins are unstable and prone to degradation during storage, resulting in loss of biological activity. Degradation may be physical in nature, including aggregation, precipitation, or gel formation. Degradation may also be chemical in nature, including hydrolytic cleavage, deamidation, cyclic imide formation, aspartic / glutamic acid isomerization, or oxidation.

[0005] The rate of degradation process increases with increasing temperature, and protein therapeutic molecules are generally more stable at lower temperatures. However, it is often difficult to develop therapeutic protein products that are stable in liquid form for their intended shelf life (usually 24 months) even under refrigeration. In addition, to ensure patient convenience, it is often necessary to develop products that are stable at high temperatures, such as up to 25°C or up to 30°C, either for a certain period or for their entire shelf life.

[0006] One of the most important parameters controlling the stability of protein therapeutics is pH. Thus, pH optimization is a key step in formulation development. Many therapeutic proteins are formulated at a selected pH between 4.0 and 8.5. It is considered important to ensure that the pH is maintained at the selected value and to ensure that pH fluctuations are minimized. Thus, it is understood that some degree of buffering capacity is required in the formulation. Larger protein molecules usually have some self-buffering capacity due to the presence of ionizable groups in the amino acid side chains of the polypeptide backbone.

[0007] The present invention addresses the problem of instability of engineered protein constructs containing an Fc domain in aqueous solutions compositions.

[0008] WO2006 / 138181A2 (Amgen) discloses self-buffering protein formulations that are substantially free of other buffering agents.

[0009] WO2009 / 073569 (Abbott) discloses aqueous formulations of antibodies, such as adalimumab, having a conductivity of less than about 2.5 mS / cm.

[0010] WO2008 / 084237 (Arecor) discloses protein compositions that do not contain significant amounts of conventional buffers. Instead, they contain a pK at least one unit lower or one unit higher than the pH of the composition. a In the present invention, a "displacement buffer" is utilized which is an additive having value.

[0011] WO2018 / 094316 (Just Biotherapeutics) discloses an eye drop formulation containing aflibercept.

[0012] WO2013 / 059412 and WO2014 / 011629 (Coherus) disclose aqueous formulations of etanercept. Summary of the Invention

[0013] (Summary of the invention) According to the present invention, there is provided an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality regulator Includes; The aqueous solution composition is provided, wherein the buffer is present in the composition at a total concentration in the range of about 0 mM to about 10 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention Described herein are stable aqueous solution compositions of engineered protein constructs containing an Fc domain in the absence or low concentration of buffer and with low ionic strength.

[0015] It should be noted that all references to "pH" herein refer to the pH of the composition evaluated at 25° C. a All references to pK of ionizable groups evaluated at 25°C. a (CRC Handbook of Chemistry and Physics, 79th Edition) th (See, DR Lide, "Eds. 1998 Edition"). If necessary, the pK a Values ​​can be estimated using a suitable calculator.

[0016] The present inventors believe that buffering agent has a detrimental effect on the stability of engineered protein constructs that contain Fc domain.Therefore, the concentration of buffering agent in the composition should be limited as much as possible.In some embodiments, a minimum amount of buffering agent is required to maintain a stable composition and minimize pH fluctuation.

[0017] When present, a buffering agent is one that has a buffering capacity at the pH of the composition. A buffering agent will usually have a pK within 1 pH unit of the pH of the composition. a However, the composition may contain an ionizable group having a pK that is one pH unit higher or lower than the pH of the composition. a Moieties having an ionizable group having a pK within 1 pH unit of the pH of the composition may also provide some buffering action if present in sufficient amounts. a In another embodiment, the (or some) buffering agent comprises an ionizable group having a pK within 1.5 pH units of the pH of the composition. a(e.g., between 1 and 1.5 pH units of the pH of the composition). In further embodiments, the (or some) buffering agent comprises an ionizable group having a pK a (eg, between 1.5 and 2 pH units of the pH of the composition).

[0018] In an embodiment, the composition is substantially free of buffer, for example, does not contain any buffer.In an embodiment, the composition contains a single buffer.In an embodiment, the composition contains two buffers.Preferably, there is more than one buffer.

[0019] In one embodiment, the total concentration of the buffer in the composition is less than 4.5 mM, e.g., less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, less than 0.5 mM, less than 0.4 mM, less than 0.3 mM, or less than 0.2 mM, or less than 0.1 mM, etc. In one embodiment, the total concentration of the buffer is in the range of about 0.1 mM to about 5 mM, e.g., about 0.5 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.5 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.5 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.5 mM to about 2 mM, about 0.1 mM to about 1 mM, or about 0.5 mM to about 1 mM, etc. In one embodiment, the total concentration of buffering agent ranges from about 1 mM to about 5 mM, about 1 mM to about 4 mM, or about 1 mM to about 3 mM. In one embodiment, the total concentration of buffering agent in the composition is <4.5 mM, such as <4 mM, <3 mM, <2 mM, <1 mM, <0.5 mM, <0.4 mM, <0.3 mM, <0.2 mM, or <0.1 mM. In one embodiment, the aqueous solution composition is substantially free of buffering agent. As used herein, "substantially free" refers to an aqueous solution composition containing less than 0.1 mM of buffering agent. When considering the concentration of buffering agent in a solution, any buffering capacity of the engineered protein construct itself should be excluded.

[0020] In one embodiment, the buffer is present in a total concentration ranging from about 1 mM to about 5 mM, for example, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM. In one embodiment, the buffer is present in the composition in a total concentration ranging from about 1.5 mM to about 5 mM, for example, about 1.5 mM to about 4 mM, about 1.5 mM to about 3 mM, or about 1.5 mM to about 2 mM. In one embodiment, the buffer is present in the composition in a total concentration ranging from about 2 mM to about 4 mM, or about 2 mM to about 3 mM. In one embodiment, the buffer is present in the composition in a total concentration ranging from about 3.5 mM to about 4 mM.

[0021] In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 10 mM, for example, about 5.5 mM to about 10 mM, about 6 mM to about 10 mM, about 6.5 mM to about 10 mM, about 7 mM to about 10 mM, about 7.5 mM to about 10 mM, about 8 mM to about 10 mM, about 8.5 mM to about 10 mM, or about 9 mM to about 10 mM, etc. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 9.5 mM, for example, about 5.5 mM to about 9.5 mM, about 6 mM to about 9.5 mM, about 6.5 mM to about 9.5 mM, about 7 mM to about 9.5 mM, about 7.5 mM to about 9.5 mM, about 8 mM to about 9.5 mM, or about 8.5 mM to about 9.5 mM, etc. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 9 mM, for example, about 5.5 mM to about 9 mM, about 6 mM to about 9 mM, about 6.5 mM to about 9 mM, about 7 mM to about 9 mM, about 7.5 mM to about 9 mM, or about 8 mM to about 9 mM, etc. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 8.5 mM, for example, about 5.5 mM to about 8.5 mM, about 6 mM to about 8.5 mM, about 6.5 mM to about 8.5 mM, about 7 mM to about 8.5 mM, or about 7.5 mM to about 8.5 mM, etc. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 8 mM, for example, about 5.5 mM to about 8 mM, about 6 mM to about 8 mM, about 6.5 mM to about 8 mM, or about 7 mM to about 8 mM. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 7.5 mM, for example, about 5.5 mM to about 7.5 mM, about 6 mM to about 7.5 mM, or about 6.5 mM to about 7.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 7 mM, for example, about 5.5 mM to about 7 mM, or about 6 mM to about 7 mM. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 6.5 mM, for example, about 5.5 mM to about 6.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 5 mM to about 6 mM.

[0022] In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 10 mM, about 3.5 mM to about 9.5 mM, about 4 mM to about 9 mM, about 4.5 mM to about 8.5 mM, about 5 mM to about 8 mM, about 5.5 mM to about 7.5 mM, or about 6 mM to about 7 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3.5 mM to about 10 mM, about 4 mM to about 10 mM, about 4.5 mM to about 10 mM, about 5 mM to about 10 mM, about 5.5 mM to about 10 mM, or about 6 mM to about 10 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 9.5 mM, about 3.5 mM to about 9.5 mM, about 4 mM to about 9.5 mM, about 4.5 mM to about 9.5 mM, about 5 mM to about 9.5 mM, about 5.5 mM to about 9.5 mM, or about 6 mM to about 9.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 9 mM, about 3.5 mM to about 9 mM, about 4 mM to about 9 mM, about 4.5 mM to about 9 mM, about 5 mM to about 9 mM, about 5.5 mM to about 9 mM, or about 6 mM to about 9 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 8.5 mM, about 3.5 mM to about 8.5 mM, about 4 mM to about 8.5 mM, about 4.5 mM to about 8.5 mM, about 5 mM to about 8.5 mM, about 5.5 mM to about 8.5 mM, or about 6 mM to about 8.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 8 mM, about 3.5 mM to about 8 mM, about 4 mM to about 8 mM, about 4.5 mM to about 8 mM, about 5 mM to about 8 mM, about 5.5 mM to about 8 mM, or about 6 mM to about 8 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 7.5 mM, about 3.5 mM to about 7.5 mM, about 4 mM to about 7.5 mM, about 4.5 mM to about 7.5 mM, about 5 mM to about 7.5 mM, about 5.5 mM to about 7.5 mM, or about 6 mM to about 7.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 7 mM, about 3.5 mM to about 7 mM, about 4 mM to about 7 mM, about 4.5 mM to about 7 mM, about 5 mM to about 7 mM, about 5.5 mM to about 7 mM, or about 6 mM to about 7 mM. In one embodiment, the buffer is present at a total concentration ranging from about 3 mM to about 6.5 mM, from about 3.5 mM to about 6.5 mM, from about 4 mM to about 6.5 mM, from about 4.5 mM to about 6.5 mM, from about 5 mM to about 6.5 mM, or from about 5.5 mM to about 6.5 mM.In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 6 mM, about 3.5 mM to about 6 mM, about 4 mM to about 6 mM, about 4.5 mM to about 6 mM, or about 5 mM to about 6 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 5.5 mM, about 3.5 mM to about 5.5 mM, about 4 mM to about 5.5 mM, or about 4.5 mM to about 5.5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 5 mM, about 3.5 mM to about 5 mM, or about 4 mM to about 5 mM. In one embodiment, the buffer is present at a total concentration in the range of about 3 mM to about 4.5 mM, or about 3.5 mM to about 4.5 mM.

[0023] The pH of an aqueous solution decreases when an acid is added and increases when a base is added. At a given temperature and atmospheric pressure, the magnitude of the pH decrease upon addition of an acid or increase upon addition of a base depends on (1) the amount of acid or base added, (2) the initial pH of the aqueous solution (i.e., before the addition of the acid or base), and (3) the presence of a buffer. Thus, (1) starting from a given pH, the addition of a larger amount of acid or base results in a larger magnitude of pH change, (2) the addition of a given amount of acid or base results in a maximum pH change at a neutral pH (i.e., pH 7.0), and the magnitude of pH change decreases as the initial pH moves away from pH 7.0, and (3) starting from a given pH, the magnitude of pH change is less in the presence of a buffer than in the absence of a buffer. Thus, a buffer has the ability to reduce the change in pH when an acid or base is added to a solution.

[0024] Suitably, a substance is considered to be a buffer if it is capable of reducing the magnitude of the change in pH of a solution by up to 75%, preferably by 50%, and most preferably by 25%, when a strong acid or base is added resulting in a 0.1 mM increase in the acid or base in the solution, compared to the same solution without the buffer.

[0025] Conversely, suitably a substance is not considered to be a buffer unless it is capable of reducing the magnitude of the change in pH of a solution by up to 75%, preferably by 50%, and most preferably by 25%, when the addition of either a strong acid or a strong base results in a 0.1 mM increase in the acid or base in the solution, compared to the same solution without the substance.

[0026] In one embodiment, the or a buffering agent is an amino acid. In another embodiment, the or a buffering agent is not an amino acid. In an embodiment, the composition does not include the amino acids lysine, arginine, histidine, glutamic acid, and aspartic acid. In an embodiment, the composition does not include cysteine.

[0027] If present, suitable buffering agents include: citric acid, histidine, maleic acid, sulfurous acid, glyoxylic acid, aspartame, glucuronic acid, aspartic acid, glutamic acid, tartaric acid, gluconic acid, lactic acid, glycolic acid, adenine, succinic acid, ascorbic acid, benzoic acid, phenylacetic acid, gallic acid, cytosine, p-aminobenzoic acid, sorbic acid, acetic acid, propionic acid, alginic acid, uric acid, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxoethyl)amine]-2-propanediol ... [N,N-bis(2-ethanesulfonic acid), phosphoric acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof.In one embodiment, the buffer is selected from the group consisting of histidine, maleic acid, sulfurous acid, glyoxylic acid, aspartame, glucuronic acid, aspartic acid, glutamic acid, tartaric acid, gluconic acid, lactic acid, glycolic acid, adenine, succinic acid, ascorbic acid, benzoic acid, phenylacetic acid, gallic acid, cytosine, p-aminobenzoic acid, sorbic acid, acetic acid, propionic acid, alginic acid, uric acid, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxoethyl) [amino]ethanesulfonic acid, piperazine, N,N'-bis(2-ethanesulfonic acid), phosphoric acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof.In one embodiment, the buffer is selected from the group consisting of citric acid, maleic acid, sulfurous acid, glyoxylic acid, aspartame, glucuronic acid, tartaric acid, gluconic acid, lactic acid, glycolic acid, adenine, succinic acid, ascorbic acid, benzoic acid, phenylacetic acid, gallic acid, cytosine, p-aminobenzoic acid, sorbic acid, acetic acid, propionic acid, alginic acid, uric acid, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfone The amine is selected from the group consisting of amines, amines having the formula: acid, piperazine, N,N'-bis(2-ethanesulfonic acid), phosphoric acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof. In one embodiment, the buffer is selected from the group consisting of citric acid, histidine, maleic acid, tartaric acid, lactic acid, benzoic acid, acetic acid, bicarbonate, phosphoric acid, and tris(hydroxymethyl)aminomethane (TRIS), such as histidine, maleic acid, tartaric acid, lactic acid, benzoic acid, acetic acid, bicarbonate, phosphoric acid, and tris(hydroxymethyl)aminomethane (TRIS), in particular histidine, lactic acid, acetic acid, phosphoric acid, and tris(hydroxymethyl)aminomethane (TRIS). For example, the buffer is phosphoric acid. Alternatively, the buffer is tris(hydroxymethyl)aminomethane (TRIS). Alternatively, the buffer is histidine. Alternatively, the buffer is lactic acid. Alternatively, the buffer is acetic acid. Alternatively, the buffer is citric acid.

[0028] In an embodiment, the composition does not include sodium phosphate. In an embodiment, when the composition includes a phosphate buffer (e.g., sodium phosphate), suitably the concentration is less than 4.5 mM, for example, less than 4.0 mM.

[0029] The primary solvent of the compositions of the invention is water, such as water for injection. Other components of the composition (e.g., polyols) may contribute to solubilizing the engineered protein construct.

[0030] The composition includes an uncharged osmolality modifier, such as a polyol. Examples of uncharged osmolality modifiers include glycerol, 1,2-propanediol, mannitol, sorbitol, sucrose, trehalose, PEG300, and PEG400. Preferably, the uncharged osmolality modifier is selected from glycerol, mannitol, 1,2-propanediol, and sucrose. If included, the total concentration of the uncharged osmolality modifier is preferably 50-1000 mM, such as 200-500 mM, for example, about 300 mM.

[0031] The composition preferably has an osmolality that is physiologically acceptable and therefore suitable for parenteral administration, and thus preferably has an osmolality in the range of about 200 mOsm / L to about 600 mOsm / L, for example, about 200 mOsm / L to about 500 mOsm / L, about 200 mOsm / L to about 400 mOsm / L, or about 300 mOsm / L.

[0032] In one embodiment, the osmolality of the composition ranges from about 200 mOsm / L to about 550 mOsm / L, e.g., from about 200 mOsm / L to about 500 mOsm / L, from about 200 mOsm / L to about 450 mOsm / L, from about 200 mOsm / L to about 400 mOsm / L, from about 200 mOsm / L to about 350 mOsm / L, or from about 200 mOsm / L to about 300 mOsm / L. In one embodiment, the osmolality of the composition is in the range of about 250 mOsm / L to about 600 mOsm / L, for example, about 300 mOsm / L to about 600 mOsm / L, about 350 mOsm / L to about 600 mOsm / L, about 400 mOsm / L to about 600 mOsm / L, about 450 mOsm / L to about 600 mOsm / L, or about 500 mOsm / L to about 600 mOsm / L. The composition is, for example, isotonic with human plasma. The composition may also be hypotonic or hypertonic, for example, intended for dilution before administration. In one embodiment, the composition is slightly hypertonic. In one embodiment, the osmolality of the composition ranges from about 300 mOsm / L to about 500 mOsm / L, such as from about 350 mOsm / L to about 500 mOsm / L, such as from about 400 mOsm / L to about 500 mOsm / L.

[0033] The composition may optionally comprise one or more neutral amino acids.As used herein, a neutral amino acid is an amino acid whose side chain does not contain ionizable groups that are significantly ionized at the pH of the composition (e.g., more than 20% of the side chain, particularly more than 50% of the side chain has negative or positive charge).Exemplary neutral amino acids are selected from glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine, particularly their L-isomers.

[0034] Preferably, the neutral amino acid is selected from glycine, methionine, proline and alanine, in particular from proline and glycine, especially proline.

[0035] When present, the total concentration of the one or more neutral amino acids may be, for example, 20 to 250 mM, such as 20 to 200 mM, for example, 50 to 150 mM, for example, 50 to 100 mM, or 25 to 75 mM. Alternatively, it may be 100 to 250 mM, for example, 150 to 200 mM.

[0036] The inventors believe that the presence of ions has a detrimental effect on the stability of engineered protein constructs containing Fc domains, therefore the ionic strength of the composition should be limited as much as possible.

[0037] The total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM, preferably less than 10 mM, e.g., less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, or less than 5 mM. The term "total ionic strength" is used herein as a function of the concentration of all ions in a solution as follows:

number

[0038] The pH of the composition is in the range of about 4.0 to about 8.5, such as about 4.0 to about 7.5 or about 5.0 to about 8.5, for example, about 6.0 to about 8.5, such as about 6.5 to about 8.5 or about 6.0 to about 7.5, for example, about 7.0 to about 7.5, etc. Other ranges of interest include about 5.0 to about 8.0, such as about 5.0 to about 7.5, for example, about 5.5 to about 7.5, in particular about 6.0 to about 7.5.

[0039] In one embodiment, the pH of the composition is in the range of about 4.0 to about 8.5, for example, about 4.0 to about 8.0, about 4.0 to about 7.5, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 4.0 to about 5.5, or about 4.0 to about 5.0, etc. In one embodiment, the pH of the composition is in the range of about 4.5 to about 8.5, for example, about 4.5 to about 8.0, about 4.5 to about 7.5, about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, or about 4.5 to about 5.5, etc. In one embodiment, the pH of the composition is in the range of about 5.0 to about 8.5, for example, about 5.0 to about 8.0, about 5.0 to about 7.5, about 5.0 to about 7.0, about 5.0 to about 6.5, or about 5.0 to about 6.0. In one embodiment, the pH of the composition is in the range of about 5.5 to about 8.5, for example, about 5.5 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, or about 5.5 to about 6.5. In one embodiment, the pH of the composition is in the range of about 6.0 to about 8.5, for example, about 6.0 to about 7.5, or about 6.0 to about 7.0.

[0040] The compositions of the invention include engineered protein constructs. Engineered protein constructs are non-natural proteins that are usually produced as products of generic engineering (gene fusion) or synthetic chemistry. Engineered protein constructs in some cases combine multiple beneficial properties originally present in two or more individual proteins (and / or individual genes encoding the individual proteins) in one intact protein construct. For example, an Fc domain can be linked (i.e., fused) to a protein with a particular desired biological function (e.g., a GLP-1 agonist), thereby protecting it from enzymatic degradation and thus increasing its circulating half-life. Alternatively, two or more antigen-binding immunoglobulin domains can be linked (i.e., fused) to the Fc domain, either directly or by additional domains, to generate engineered antibodies with multiple valencies and / or specificities. Such artificial manipulation of protein structures using principles of genetic engineering or synthetic chemistry, while often resulting in engineered constructs with highly desirable physiological properties for disease treatment, often leads to non-native exposure of structural motifs at the newly generated protein surface, such as, for example, extensive hydrophobic patches or other instability hotspots that are not normally exposed. This in turn leads to compromised stability of the engineered protein constructs, such as increased tendency to aggregate. The present invention addresses such increased instability of engineered protein constructs.

[0041] Fully human monospecific antibodies (and natural antibodies of other non-human species), even when produced by expression in a heterologous host such as bacteria or fungi, are not encompassed by the term "engineered protein construct". Human monospecific antibodies produced by non-human animals (such as mice) engineered to have a human immune system are also not encompassed by the term "engineered protein construct". For example, adalimumab is not an engineered protein construct. In one embodiment, the engineered protein construct is not a chimeric antibody, in particular a monospecific chimeric antibody. In one embodiment, the engineered protein construct is not a humanized antibody, in particular a monospecific humanized antibody.

[0042] The engineered protein constructs of the invention comprise an Fc domain. An Fc domain is a domain of an antibody, including derivatives thereof, that interacts with an Fc receptor or a protein that is part of the complement system to activate the immune system. The Fc domain can be derived, for example, from IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, IgY, and IgE isotypes. Fc domains from IgG, IgA, and IgD isotypes comprise two identical protein chain fragments connected by disulfide bonds, each derived from the second and third constant domains of the antibody heavy chain. Fc domains from IgM and IgE isotypes comprise three identical protein chain fragments connected by disulfide bonds, each derived from the second, third, and fourth constant domains of the antibody heavy chain. The Fc domain may be optionally glycosylated. Most preferably, the Fc domain is an IgG, particularly an IgG1 or IgG4, especially an IgG1 Fc domain. The Fc domain may typically have a molecular weight of 25-40 kDa, which may be higher in the case of glycosylated Fc domains. Derivatives of Fc domains encompassed by the term include domains known as Fcabs, in which the Fc domain has been modified to include an antigen-binding site (see Protein Engineering, Design and Selection (2017) 30(9) 657-671). Further examples of derivatives include conjugated derivatives, such as engineered protein constructs that include an Fc domain conjugated to another moiety. Such moieties include chemically inert polymers such as PEG. In some embodiments, the Fc domain contains one or more modifications that alter one or more properties of the engineered protein construct, such as serum half-life, complement binding, Fc receptor binding, and / or effector function (e.g., antigen-dependent cytotoxicity).

[0043] In an embodiment, the engineered protein construct is a fusion of the Fc domain with a heterologous polypeptide. One, and preferably both, chains of the Fc domain are linked (i.e. fused) to a heterologous polypeptide. A heterologous polypeptide is a polypeptide that is not naturally found within the Fc domain or adjacent sequences thereof, and in particular is not the antigen-binding portion of an antibody (i.e. the Fab portion). Suitably, each chain of the Fc domain is linked (i.e. fused) to the same heterologous polypeptide such that the engineered protein construct is a homodimer.

[0044] In an embodiment, the heterologous polypeptide can bind to a ligand, preferably a specific ligand. The heterologous polypeptide may be capable of interacting with another protein, such as a protein that has a role in the human body, including but not limited to a cytokine. In an embodiment, the heterologous polypeptide is selected from a cytokine, a growth factor, a clotting factor, an enzyme, a receptor protein, a GLP-1 agonist, and functional fragments and domains thereof.

[0045] In an embodiment, the heterologous polypeptide can bind to tumor necrosis factor (TNF), e.g., TNFα, and can include, e.g., a TNF receptor, e.g., TNF receptor 2, particularly a soluble form thereof. In an embodiment, the heterologous polypeptide can bind to a membrane protein, such as CD80 or CD86, and can include, e.g., the extracellular domain of CLTA-4 or a portion thereof. In an embodiment, the heterologous polypeptide can bind to VEGF, and can include, e.g., the extracellular domain of VEGFR1 and / or VEGR2 or a portion thereof. In an embodiment, the heterologous polypeptide can bind to IL-1, and can include, e.g., the extracellular domain of an interleukin, such as IL-1R11 and / or IL-1RAcP, or a portion thereof. In an embodiment, the heterologous polypeptide can bind to a thrombopoietin receptor, such as c-Mpl. In an embodiment, the heterologous polypeptide is a clotting factor, such as factor VIII or factor IX, or a portion thereof. In an embodiment, the heterologous polypeptide is a hActRIIb protein or a derivative thereof. In an embodiment, the heterologous polypeptide is a protease inhibitor. In an embodiment, the heterologous polypeptide is a GLP-1 agonist.

[0046] Exemplary engineered protein constructs containing Fc domain include etanercept, abatacept, belatacept, aflibercept, rilonacept, romiplostim, eloctate, luspatercept, dulaglutide and orprolix.In one embodiment, engineered protein construct is dulaglutide.In one embodiment, engineered protein construct is abatacept.In one embodiment, engineered protein construct is aflibercept.In one embodiment, engineered protein construct is etanercept.

[0047] In an embodiment, the engineered protein construct just comprises the antigen-binding portion of an antibody (i.e., the Fab portion). In an embodiment, the engineered protein construct is a bispecific antibody in the format of a four-chain antibody with two different variable binding regions. In an embodiment, the engineered protein construct is a bispecific antibody in the format of a two-chain antibody with two different variable binding regions (i.e., a heavy chain-only antibody). A heavy chain-only antibody can be derived, for example, from an antibody isolated from a camelid. Whether in a two-chain or four-chain format, such a bispecific antibody can have, for example, a set of three CDRs for arms a and b of the antibody, designated CDR1a, CDR2a, CDR3a, CDR1b, CDR2b, and CDR3b, where CDR1a is not the same as CDR1b, and / or CDR2a is not the same as CDR2b, and / or CDR3a is not the same as CDR3b. Suitably, none of the six CDRs is identical to any other of the six CDRs.

[0048] In an embodiment, said engineered protein construct is a bispecific or trispecific antibody in the Fcab format, in which the Fc domain has been modified to comprise an antigen binding site.

[0049] In certain embodiments, two engineered protein constructs can associate, for example, via disulfide bonds to form a dimeric protein.

[0050] In embodiments, the engineered protein construct comprises an IgG Fc domain and two or more (e.g., two, three, four, five, or six, such as, for example, two or four) immunoglobulin chain variable domains. As used herein, an immunoglobulin chain variable domain is an antigen-binding domain derived from an antibody. An immunoglobulin chain variable domain can be directly linked to the Fc domain or can be indirectly linked to the Fc domain, for example, via an intervening constant domain. The specificity of an immunoglobulin chain variable domain is determined by the CDRs, and an immunoglobulin chain variable domain typically has three CDRs. Exemplary immunoglobulin chain variable domains include the V CDRs from conventional four-chain antibodies, and the V CDRs from conventional four-chain antibodies. H and V L domains, as well as V domains derived from heavy chain-only antibodies, e.g., those found in camelids. HHIn an embodiment, the engineered protein construct includes an IgG Fc domain formed of two chains, each of which is directly or indirectly linked to one or more (e.g., one, two, or three, such as one or two) immunoglobulin chain variable domains. In an embodiment, the engineered protein construct includes an IgG Fc domain formed of two chains, each of which is directly linked to one or more (e.g., one or two) immunoglobulin chain variable domains. As used herein, "directly linked" means directly linked (i.e., fused), optionally via a peptide linker, and without any intervening domain that is not an immunoglobulin chain variable domain (e.g., a constant domain). In an embodiment, the engineered protein construct includes an IgG Fc domain formed of two chains, each of which is indirectly linked to one or more (e.g., one or two) immunoglobulin chain variable domains. As used herein, "indirectly linked" means linked (i.e., fused) at one or more intervening domains (e.g., a constant domain). Peptide linkers may be present between the various domains. In an embodiment, each of the immunoglobulin chain variable domains has the same specificity (i.e., the engineered protein construct is monospecific), e.g., each of the immunoglobulin chain variable domains is the same. In an embodiment, the construct comprises two or more (e.g., two) immunoglobulin chain variable domains with different specificities (i.e., the engineered protein construct is multispecific, e.g., bispecific), e.g., at least two of the immunoglobulin chain variable domains are not the same.

[0051] The engineered protein construct is preferably a therapeutic engineered protein construct. Such engineered protein constructs have a desired therapeutic or prophylactic activity and are indicated for the treatment, inhibition, or prevention of a disease or medical disorder. In some embodiments, the engineered protein construct is substantially pure, i.e., the composition comprises a single engineered protein construct and does not contain substantial amounts of any additional proteins. In a preferred embodiment, the engineered protein construct comprises at least 99%, preferably at least 99.5%, more preferably at least about 99.9% of the total protein content of the composition. In a preferred embodiment, the engineered protein construct is sufficiently pure for use in a pharmaceutical composition.

[0052] The engineered protein construct is preferably present in the composition at a concentration of about 1-400 mg / ml, preferably 10-200 mg / ml, more preferably 20-100 mg / ml, for example about 50 mg / ml.

[0053] The composition may include a non-ionic surfactant, which may be selected from the group consisting of polysorbates, alkyl glycosides, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol, and alkyl phenyl ethers of polyethylene glycol.

[0054] A particularly suitable class of nonionic surfactants are the polysorbates (fatty acid esters of ethoxylated sorbitan), such as polysorbate 20 or polysorbate 80. Polysorbate 20 is a monoester formed from lauric acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 80 is a monoester formed from oleic acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 20 is known under various brand names, including, among others, Tween 20 and also Alkest TW 20. Polysorbate 80 is known under various brand names, including, among others, Tween 80 and also Alkest TW 80. Other suitable polysorbates include polysorbate 40 and polysorbate 60.

[0055] Another suitable class of nonionic surfactants are the alkyl glycosides, especially dodecyl maltoside.Other alkyl glycosides include dodecyl glucoside, octyl glucoside, octyl maltoside, decyl glucoside, decyl maltoside, tridecyl glucoside, tridecyl maltoside, tetradecyl glucoside, tetradecyl maltoside, hexadecyl glucoside, hexadecyl maltoside, sucrose monooctanoate, sucrose monodecanoate, sucrose monododecanoate, sucrose monotridecanoate, sucrose monotetradecanoate, and sucrose monohexadecanoate.

[0056] Another suitable class of nonionic surfactants are alkyl ethers of polyethylene glycols, particularly those known under the brand name Brij, such as those selected from polyethylene glycol (2) hexadecyl ether (Brij 52), polyethylene glycol (2) oleyl ether (Brij 93), and polyethylene glycol (2) dodecyl ether (Brij L4). Other suitable Brij surfactants include polyethylene glycol (4) lauryl ether (Brij 30), polyethylene glycol (10) lauryl ether (Brij 35), polyethylene glycol (20) hexadecyl ether (Brij 58), and polyethylene glycol (10) stearyl ether (Brij 78).

[0057] Another suitable class of nonionic surfactants are the poloxamers, particularly block copolymers of polyethylene glycol and polypropylene glycol, also known as poloxamer 188, poloxamer 407, poloxamer 171, and poloxamer 185. Poloxamers are also known under the brand names Pluronics or Koliphors. For example, poloxamer 188 is sold as Pluronic F-68.

[0058] Another suitable class of non-ionic surfactants are the alkyl phenyl ethers of polyethylene glycol, particularly 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, also known under the brand name Triton X-100.

[0059] In one embodiment, the nonionic surfactant is a polysorbate or a poloxamer, preferably a polysorbate. The concentration of the nonionic surfactant in the composition will usually be within the range of 10 to 2000 μg / ml, for example, 50 to 1000 μg / ml, 100 to 500 μg / ml, or about 200 μg / ml.

[0060] Further, the composition of the present invention may contain a preservative, such as a phenol-based preservative or a benzyl-based preservative. The preservative is preferably selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, and methylparaben, in particular phenol, m-cresol, and benzyl alcohol. The concentration of the preservative is usually 10-100 mM, such as 20-80 mM, for example 25-50 mM. The optimal concentration of the preservative in the composition is determined by whether the composition meets the Pharmacopoeia Antimicrobial Effectiveness Test (USP). <51> , Vol. 32) are selected to ensure that they pass the exam.

[0061] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; one or more neutral amino acids; and Uncharged osmolality regulator Includes: The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration in the composition of about 0.1 to about 5 mM, such as about 1 to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as about 5.5 mM to about 8 mM.

[0062] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 6.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 4.0 to about 9.5, for example, about 5.0 to about 9.5, and the pK a one or more buffering agents that are substances having at least one ionizable group that is within 2 pH units, e.g., within 1.5 pH units, e.g., within 1 pH unit, of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality regulator Includes: The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as from about 5.5 mM to about 8 mM.

[0063] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain that are bispecific antibodies in the form of two-chain antibodies with two different variable binding regions; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents that are substances having at least one ionizable group that is within 2 pH units, e.g., within 1.5 pH units, e.g., within 1 pH unit, of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality modifiers, e.g., polyols Includes; The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 0 mM to about 5 mM, such as from about 1 mM to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as from about 5.5 mM to about 8 mM.

[0064] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; one or more neutral amino acids; Uncharged osmolality modifiers, such as polyols; and Nonionic Surfactants Includes; The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as from about 5.5 mM to about 8 mM.

[0065] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK ais in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; For example, one or more neutral amino acids selected from glycine, methionine, proline, and alanine; Uncharged osmolality modifiers, such as polyols; and preservative Includes The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as from about 5.5 mM to about 8 mM.

[0066] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: an engineered protein construct comprising an Fc domain, which is a fusion of the Fc domain with a heterologous protein selected from a cytokine, a growth factor, a clotting factor, an enzyme, a receptor protein, a GLP-1 agonist, and functional fragments and domains thereof; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality modifiers, e.g., polyols Includes; The aqueous solution composition is provided wherein the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM. In one specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM. In another specific subembodiment, the one or more buffering agents are present in the composition at a total concentration ranging from about 5.5 mM to about 10 mM, such as from about 5.5 mM to about 8 mM.

[0067] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: an engineered protein construct containing an Fc domain, which is a bispecific antibody in the format of a four-chain antibody with two different variable binding regions; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pK a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality modifiers, e.g., polyols Includes; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM. In a specific subembodiment, one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 to about 3 mM.

[0068] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain that are bispecific antibodies in the form of two-chain antibodies with two different variable binding regions; Optionally, pK a is in the range of about 3.0 to about 9.5, and the pKa one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality modifiers, e.g., polyols Includes; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM. In a specific subembodiment, one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM.

[0069] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 6.0 to about 7.5, comprising: engineered protein constructs containing an Fc domain that are bispecific antibodies in the form of two-chain antibodies with two different variable binding regions; Optionally, pK a is in the range of about 5.0 to about 8.5, and the pK a one or more buffering agents that are substances having at least one ionizable group, the pH of which is within 2 pH units, e.g., within 1.5 pH units, e.g., within 1 pH unit, of the pH of the composition; Optionally, one or more neutral amino acids; and Uncharged osmolality modifiers, e.g., polyols Includes; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM. In a specific subembodiment, one or more buffering agents are present in the composition at a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 1 mM to about 3 mM.

[0070] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 6.0 to about 8.5, for example, about 6.5 to about 8.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphoric acid and tris(hydroxymethyl)aminomethane (TRIS); One or more neutral amino acids selected from, for example, proline and glycine; and Uncharged osmolality modifiers, e.g., sucrose Includes; the buffering agent is present in the composition at a total concentration in the range of about 0.1 mM to about 5 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM.

[0071] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 6.0 to about 8.5, for example, about 6.5 to about 8.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphoric acid and tris(hydroxymethyl)aminomethane (TRIS); One or more neutral amino acids selected from, for example, proline and glycine; and Uncharged osmolality modifiers, e.g., sucrose Includes; The aqueous solution composition is provided wherein the buffer is present in the composition at a total concentration in the range of about 5.5 mM to about 10 mM; and the total ionic strength of the composition excluding the contribution of the engineered protein construct is less than 20 mM, and preferably less than 10 mM.

[0072] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 5.0 to about 8.0, for example, about 5.0 to about 7.5, for example, about 5.5 to about 7.5, for example, about 6.0 to about 7.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphate and citrate; and Uncharged osmolality modifiers, e.g., sucrose Includes; the buffering agent is present in the composition at a total concentration in the range of about 0.1 mM to about 5 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM.

[0073] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 5.0 to about 8.0, for example, about 5.0 to about 7.5, for example, about 5.5 to about 7.5, for example, about 6.0 to about 7.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphate and citrate; and Uncharged osmolality modifiers, e.g., sucrose Includes; the buffering agent is present in the composition at a total concentration in the range of about 5.5 mM to about 10 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM, preferably less than 10 mM.

[0074] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 5.0 to about 8.0, for example, about 5.0 to about 7.5, for example, about 5.5 to about 7.5, for example, about 6.0 to about 7.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphate and citrate; One or more neutral amino acids selected from, for example, proline and glycine; and Uncharged osmolality modifiers, e.g., sucrose Includes; the buffering agent is present in the composition at a total concentration in the range of about 0.1 mM to about 5 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM.

[0075] In one embodiment, the present invention provides an aqueous solution composition having a pH range of about 5.0 to about 8.0, for example, about 5.0 to about 7.5, for example, about 5.5 to about 7.5, for example, about 6.0 to about 7.5, comprising: engineered protein constructs containing an Fc domain; a buffering agent selected from phosphate and citrate; One or more neutral amino acids selected from, for example, proline and glycine; and Uncharged osmolality modifiers, e.g., sucrose Includes; the buffering agent is present in the composition at a total concentration in the range of about 5.5 mM to about 10 mM; and the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM, preferably less than 10 mM.

[0076] In one embodiment, the present invention provides 1. An aqueous solution composition having a pH range of about 4.0 to about 8.5, comprising: an engineered protein construct comprising an IgG Fc domain and two or more immunoglobulin chain variable domains; pK a is in the range of about 3.0 to about 9.5, and the pK a is a substance having at least one ionizable group that is within 2 pH units of the pH of the composition, and one or more buffers selected from, e.g., citric acid, histidine, maleic acid, tartaric acid, lactic acid, benzoic acid, acetic acid, bicarbonate, phosphate, and tris(hydroxymethyl)aminomethane (TRIS); Non-ionic surfactants; an uncharged osmolality modifier selected from glycerol, 1,2-propanediol, mannitol, sorbitol, sucrose, trehalose, PEG300, and PEG400; and Optionally, one or more neutral amino acids Includes; The aqueous solution composition is provided wherein the buffer is present in the composition at a total concentration of about 1 mM to about 10 mM; the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than about 20 mM (e.g., less than about 10 mM); and the osmolality of the composition is in the range of about 200 mOsm / L to about 500 mOsm / L, such as about 350 mOsm / L to about 500 mOsm / L.

[0077] Suitably, the compositions of the present invention remain a clear solution after storage at 2-8° C. for an extended period of time, such as at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0078] Suitably, the compositions of the present invention remain a clear solution after storage at 25° C. for an extended period of time, such as at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0079] Suitably, the compositions of the present invention remain a clear solution after storage at 30° C. for an extended period of time, such as at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0080] Suitably, the compositions of the invention remain a clear solution after storage at 40° C. or 50° C. (i.e., temperatures suitable for accelerated stability testing) for a period of time, such as at least 1 day, 3 days, 1 week, 2 weeks, or 4 weeks.

[0081] Suitably, the compositions of the invention have improved storage stability at either 2-8° C. or elevated temperatures than in comparable compositions containing higher concentrations of the same buffer(s).

[0082] Suitably, the compositions of the present invention have improved storage stability at either 2-8° C. or elevated temperatures than in comparable compositions having higher total ionic strength.

[0083] In one embodiment, the composition of the invention contains no more than 5% total impurities, such as no more than 4%, such as no more than 3%, such as no more than 2% total impurities (by total weight of engineered protein constructs in the composition as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) after storage at 2-8°C for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0084] In one embodiment, the composition of the invention contains no more than 5% total impurities, such as no more than 4%, such as no more than 3%, such as no more than 2% total impurities (by total weight of engineered protein constructs in the composition as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) after storage at 25° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0085] In one embodiment, the composition of the invention contains no more than 5% total impurities, such as no more than 4%, such as no more than 3%, such as no more than 2% total impurities (by total weight of engineered protein constructs in the composition as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) after storage at 30° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0086] In one embodiment, the composition of the invention contains no more than 5% total impurities, such as no more than 4%, such as no more than 3%, such as no more than 2% total impurities (by total weight of engineered protein constructs in the composition as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) after storage at 40° C. for at least 1 day, 3 days, 1 week, 2 weeks, or 4 weeks.

[0087] In one embodiment, the compositions of the invention contain lower levels of impurities (as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) than a commercial composition containing the same pharmaceutical ingredient (as measured by the same technique) after storage at 2-8° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0088] In one embodiment, the compositions of the invention contain lower levels of impurities (as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) than a commercial composition containing the same pharmaceutical ingredient (as measured by the same technique) after storage at 25° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0089] In one embodiment, the compositions of the invention contain lower levels of impurities (as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable technique) than a commercial composition containing the same pharmaceutical ingredient (as measured by the same technique) after storage at 30° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0090] In one embodiment, the compositions of the invention contain lower levels of impurities (as measured by cation exchange chromatography, size exclusion chromatography, or similar suitable techniques) after storage at 40° C. or 50° C. for at least 1 day, 3 days, 1 week, 2 weeks, or 4 weeks than a commercial composition containing the same pharmaceutical ingredient (as measured by the same techniques).

[0091] In an embodiment, the composition of the present invention is a composition for use in therapy.In an embodiment, the composition of the present invention is a pharmaceutical composition.Compositions, such as those intended for intravenous administration, can be prepared as concentrates for dilution before administration.

[0092] All the embodiments described above with respect to the aqueous solution composition apply equally to the method and use of the present invention.

[0093] Also provided are containers, e.g., made of plastic or glass, that contain a single dose or multiple doses of the compositions described herein. The containers can be, for example, vials, pre-filled syringes, pre-filled infusion bags, or cartridges designed to be replaceable items for use with injection devices.

[0094] Suitably, the compositions of the invention may be packaged for injection, particularly for intravenous infusion, intravenous injection, subcutaneous injection, or intramuscular injection.

[0095] One aspect of the present invention is an injection or infusion device for single or multiple use, in particular a device suitable for subcutaneous or intramuscular injection or infusion, comprising a container with one or multiple doses of the composition of the present invention together with an injection needle.In an embodiment, the container is a replaceable cartridge with multiple doses.In an embodiment, the injection device is in the form of a pen.In an embodiment, the injection device is in the form of a pre-filled syringe.In an embodiment, the injection or infusion device is in the form of a pump or another wearable injection or infusion device.

[0096] Compositions according to the present invention, as described herein, are expected to have good physical and chemical stability. EXAMPLES

[0097] (Example) (General method) Methods for assessing the stability of protein constructs ((a) Visual evaluation) Visible particles are preferably detected using 2.9.20. European Pharmacopoeia Monograph (Particulate Contamination: Visible Particles). The equipment required is: A matte black panel of suitable size held vertically A non-reflective white panel of suitable size held vertically next to the black panel An adjustable lamp holder fitted with a white light source with a suitable shade and a suitable diffuser (an observation illuminator containing two 13 W fluorescent tubes, each 525 mm long, is suitable). The intensity of the illumination at the observation point is maintained between 2000 lux and 3750 lux.

[0098] Any attached labels are removed from the container and the exterior is cleaned and dried. The container is gently rotated or inverted to ensure that no air bubbles are introduced and observed in front of a white panel for approximately 5 seconds. The procedure is repeated in front of a black panel. The presence of particles is noted. The visual scores are ranked as follows: Visual score A: Clear solution virtually free of particles, <10 particles Visual score B: Particles visible only under lamp Visual score C: Significant visible change in appearance under normal laboratory conditions. Particles in samples with a visual score of C are clearly detectable in an informal visual evaluation under normal light, whereas samples with visual scores of A and B generally appear as clear solutions in the same evaluation. Samples with visual scores of A and B are considered "passes"; samples with a visual score of C are considered "fails."

[0099] (b) Size Exclusion Chromatography (SEC) The amount of high molecular weight species is measured using a 300 x 7.8 mm TSK Gel G3000 SWXL (or equivalent) size exclusion column with a guard column. The mobile phase is sodium phosphate buffer pH 6.75, with a flow rate of 1 ml / min, an injection volume of 20 μl, and detection at 280 nm. The results are expressed as % High Molecular Weight Species (HMWS), i.e., the sum of all peak areas corresponding to aggregated proteins / sum of all protein-related peaks in the chromatogram. Small variations between time points can be observed in terms of absolute values ​​of % areas (monomer, HMWS, and low molecular weight species (LMWS)), for example due to the use of repeated size exclusion columns. However, within a given time point, the samples are tested with the same condition of the column, so the values ​​obtained within that time point represent a very good indication of the relative stability of the protein in the aqueous solution tested. An increase in %HMWS refers to the change in %HMWS observed at a given time point compared to the %HMWS value at time zero (ie, immediately prior to incubation at storage temperature).

[0100] (c) Cation exchange chromatography (CEX) The amount of related species is measured using a Protein-Pak Hi Res SP column. Mobile phase A is 20 mM sodium phosphate, pH 6.5; mobile phase B is 20 mM sodium phosphate + 0.5 M NaCl, pH 6.0. The following gradient elution is used: 0 min - 100% A, 4 min - 80% A, 10 min - 55% A, 12 min - 0% A. The flow rate is 1.0 ml / min; the injection volume is 3 μl with UV detection at 214 nm. The results are expressed as % main peak (i.e. native protein), % acidic species, and % basic species. % related species = % acidic species + % basic species.

[0101] Example 1 - Example Formulation The following example formulations may be prepared: Example A Fc-fusion protein* 50mg / ml Sodium phosphate 1mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0102] Example B Fc-fusion protein* 50mg / ml Sodium acetate 3mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 5.0 using either hydrochloric acid or sodium hydroxide.

[0103] Example C Fc-fusion protein* 50mg / ml Sodium acetate 3mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 5.0 using either hydrochloric acid or sodium hydroxide.

[0104] Example D Fc-fusion protein* 50mg / ml Sodium phosphate 3mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0105] Example E Fc-fusion protein* 50mg / ml TRIS 1mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0106] Example F Fc-fusion protein* 50mg / ml TRIS 3mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0107] Example G Fc-fusion protein* 50mg / ml TRIS 3mM Sucrose 500mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0108] Example H Fc-fusion protein* 50mg / ml Sodium phosphate 1mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0109] Example I Fc-fusion protein* 50mg / ml TRIS 1mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0110] Example J Fc-fusion protein* 50mg / ml Sodium phosphate 1mM Sucrose 300mM Glycine 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0111] Example K Fc-fusion protein* 50mg / ml Sodium phosphate 1mM Sucrose 400mM Glycine 150mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0112] Example L Fc-fusion protein* 50mg / ml TRIS 1mM Sucrose 300mM Glycine 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0113] Example M Fc-fusion protein* 50mg / ml Sodium phosphate 7mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0114] (Example N) Fc-fusion protein* 50mg / ml Sodium acetate 7mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 5.0 using either hydrochloric acid or sodium hydroxide.

[0115] (Example O) Fc-fusion protein* 50mg / ml Sodium phosphate 7mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0116] Example P Fc-fusion protein* 50mg / ml Sodium phosphate 7mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0117] Example Q Fc-fusion protein* 50mg / ml Sodium phosphate 9mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0118] (Example R) Fc-fusion protein* 50mg / ml Sodium acetate 9mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 5.0 using either hydrochloric acid or sodium hydroxide.

[0119] (Example S) Fc-fusion protein* 50mg / ml Sodium phosphate 9mM Sucrose 300mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide.

[0120] Example T Fc-fusion protein* 50mg / ml Sodium phosphate 9mM Sucrose 300mM Proline 100mM Water for injection (appropriate amount) The pH is adjusted to 7.0 using either hydrochloric acid or sodium hydroxide. *The Fc-fusion protein is (1) etanercept, (2) aflibercept, or (3) dulaglutide.

[0121] (Examples AA to TT) The same formulations as in Examples A-T are prepared, except that instead of an Fc-fusion protein, an engineered protein construct containing a bispecific antibody Fc domain is used.

[0122] (Examples AAA to TTT) The same formulations as in Examples A-T are prepared, except that instead of an Fc-fusion protein, an engineered protein construct containing an Fc domain that is a trispecific antibody is used.

[0123] The stability of the formulations is determined using visual evaluation, SEC, and CEX (see general methods) after 2, 4, and 8 weeks of incubation at 40°C.

[0124] The stability of the formulations is determined using visual evaluation, SEC, and CEX (see general methods) after 2, 4, 8, and 12 weeks of incubation at 25°C.

[0125] The stability of the formulations is determined using visual evaluation, SEC, and CEX (see general methods) after 2, 4, 8, and 12 weeks of incubation at 2-8°C.

[0126] Example 2 - Effect of Buffer Concentration and Osmotic Modifier Charge on Dulaglutide Stability at 40°C and 50°C The effect of buffer concentration and osmotic modifier charge on the stability of dulaglutide (1 mg / ml) was investigated. Citrate and phosphate buffers were tested. Sodium chloride (150 mM) was used as the charged osmotic modifier and sucrose (250 mM) was used as the uncharged osmotic modifier. All formulations tested were adjusted to pH 6.5. In addition, a formulation of a commercial dulaglutide product (Trulicity) was used as a control formulation for comparison. Table 1 summarizes the formulations tested. All formulations were stressed at 40°C and 50°C for 4 weeks. The stability of dulaglutide was followed by monitoring the rate of high molecular weight species formation using SEC.

[0127] Table 1: Formulations of dulaglutide tested. All formulations contained dulaglutide (1 mg / ml) and were adjusted to pH 6.5. [Table 1] *Prepared by mixing sodium citrate (9.3 mM) with citric acid (0.7 mM).

[0128] All formulations tested passed visual inspection after storage at both temperatures (visual score A). The HMWS formation rates in formulations 2-1 to 2-21 after storage at 50°C and 40°C are shown in Table 2. HMWS formation rates were lowest in formulations with very low buffer concentrations and uncharged osmolality modifiers. In the presence of sucrose, no detectable increase in HMWS was observed in compositions containing up to 5 mM citrate buffer at 40°C. HMWS formation rates increased at higher citrate buffer concentrations. Similarly, under more accelerated stress at 50°C, HMWS formation rates were proportional to citrate buffer concentration and were very low at buffer concentrations below 5 mM. Similar observations were made with phosphate buffer. In contrast, HMWS formation was observed at all buffer concentrations in the presence of sodium chloride.

[0129] Table 2: Stability of dulaglutide (1 mg / ml) in formulations 2-1 through 2-21 at 40° C. and 50° C. as assessed by SEC. [Table 2] *Unfortunately, testing of this formulation failed for reasons unrelated to the formulation itself.

[0130] Example 3 - Effect of Buffer Concentration and Osmotic Modifier Charge on Abatacept Stability at 50°C The effect of buffer concentration and osmotic modifier charge on the stability of abatacept (4.25 mg / ml) was investigated. Citrate and phosphate buffers were tested. Sodium chloride (150 mM) was used as the charged osmotic modifier and sucrose (250 mM) was used as the uncharged osmotic modifier. All formulations tested were adjusted to pH 6.8. In addition, a formulation of the commercial abatacept product (Orencia) was used as a control formulation for comparison. Table 3 summarizes the formulations tested. All formulations were stressed at 50° C. for 2 weeks. The stability of abatacept was followed by monitoring the rate of high molecular weight species formation using SEC.

[0131] Table 3: Abatacept formulations tested. All formulations contained abatacept (4.25 mg / ml) and were adjusted to pH 6.8. [Table 3]

[0132] All formulations tested passed the visual inspection (visual score A) after storage at 50°C. The HMWS formation rates in formulations 3-1 to 3-19 after storage at 50°C are shown in Table 4. HMWS formation rates were lowest in formulations containing very low buffer concentrations and uncharged osmolality modifiers. HMWS formation rates increased with increasing buffer concentration in both citrate and phosphate buffers. In contrast, HMWS formation was very high in the presence of sodium chloride regardless of buffer concentration.

[0133] Table 4: Stability of Abatacept (4.25 mg / ml) at 50°C in Formulations 3-1 through 3-19 as assessed by SEC. [Table 4]

[0134] Example 4 - Effect of Buffer Concentration and Osmotic Modifier Charge on Abatacept Stability at 40°C The effect of citrate buffer concentration and osmotic modifier charge on the stability of abatacept (4.25 mg / ml) was investigated. Sodium chloride (150 mM) was used as the charged osmotic modifier and sucrose (250 mM) was used as the uncharged osmotic modifier. All formulations tested were adjusted to pH 6.8. Table 5 summarizes the formulations tested. All formulations were stressed at 40° C. for 2 weeks. The stability of abatacept was followed by monitoring the rate of high molecular weight species formation using SEC.

[0135] Table 5: Abatacept formulations tested. All formulations contained abatacept (4.25 mg / ml) and were adjusted to pH 6.8. [Table 5]

[0136] All formulations tested passed visual inspection (visual score A) after storage at 40°C. The HMWS formation rates in formulations 4-1 to 4-9 after storage at 40°C are shown in Table 6. HMWS formation rates were lowest in compositions containing very low citrate buffer concentrations and uncharged osmolality modifiers. HMWS formation rates increased with increasing citrate buffer concentrations. In contrast, HMWS formation was very high in the presence of sodium chloride regardless of buffer concentration.

[0137] Table 6: Stability of Abatacept (4.25 mg / ml) in Formulations 4-1 through 4-9 at 40° C. as assessed by SEC. [Table 6]

[0138] Example 5 - Effect of Proline on Abatacept Stability at 50°C in the Presence of 1 mM Buffer and Uncharged Osmolarity Modifier The effect of proline (50 mM) on the stability of abatacept (4.25 mg / ml) was investigated in the presence of 1 mM buffer and uncharged osmolality modifier. Sucrose (250 mM) was used as the uncharged osmolality modifier. All formulations tested were adjusted to pH 6.8. Table 7 summarizes the formulations tested. All formulations were stressed at 50° C. for 2 weeks. The stability of abatacept was followed by monitoring the rate of high molecular weight species formation using SEC.

[0139] Table 7: Abatacept formulations tested. All formulations contained abatacept (4.25 mg / ml) and were adjusted to pH 6.8. [Table 7]

[0140] All formulations tested passed the visual test (visual score A) after storage at 50° C. The HMWS formation rates in formulations 5-1 through 5-4 after storage at 50° C. are shown in Table 8. The presence of proline (50 mM) in a composition containing 1 mM buffer and sucrose (250 mM) resulted in a lower HMWS formation rate.

[0141] Table 8: Stability of Abatacept (4.25 mg / ml) in Formulations 5-1 to 5-4 at 50° C. as assessed by SEC. [Table 8]

[0142] Example 6 - Effect of Proline and Glycine on Abatacept Stability at 40°C in the Presence of 1 mM Buffer and Uncharged Osmolarity Modifier The effect of proline (50 mM) and glycine (50 mM) on the stability of abatacept (4.25 mg / ml) was investigated in the presence of 1 mM buffer and uncharged osmolality modifiers. Citrate and phosphate buffers were tested. Sucrose (250 mM) was used as the uncharged osmolality modifier. All formulations tested were adjusted to pH 6.8. Table 9 summarizes the formulations tested. All formulations were stressed at 40° C. for 2 weeks. The stability of abatacept was followed by monitoring the rate of high molecular weight species formation using SEC.

[0143] Table 9: Abatacept formulations tested. All formulations contained abatacept (4.25 mg / ml) and were adjusted to pH 6.8. [Table 9]

[0144] All formulations tested passed the visual test (visual score A) after storage at 40° C. The HMWS formation rates in formulations 6-1 through 6-6 after storage at 40° C. are shown in Table 10. The presence of proline (50 mM) and glycine (50 mM) in a composition containing 1 mM buffer and sucrose (250 mM) resulted in a lower HMWS formation rate.

[0145] Table 10: Stability of Abatacept (4.25 mg / ml) in Formulations 6-1 to 6-6 at 40° C. as assessed by SEC. [Table 10]

[0146] Example 7 - Effect of buffer concentration, osmolality adjuster, and neutral amino acid on the stability of abatacept in low strength compositions at 40°C The effect of buffer concentration, osmolality modifier, and neutral amino acid on the stability of abatacept at 40°C was investigated. Citric acid was used as the buffer. Sucrose and 1,2-propanediol were tested as osmolality modifiers and proline was tested as the neutral amino acid. All formulations tested were adjusted to pH 6.8. Table 11 summarizes the formulations tested. All formulations were stressed at 40°C for 2 and 4 weeks. The stability of abatacept was followed by monitoring the rate of high molecular weight species formation using SEC.

[0147] Table 11: Abatacept formulations tested. All formulations contained abatacept (4.25 mg / ml) and were adjusted to pH 6.8. [Table 11]

[0148] All formulations tested passed visual inspection (visual score A) after storage at 40°C for 2 and 4 weeks. The HMWS formation rates in formulations 7-1 to 7-18 after storage at 40°C are shown in Table 12. It was shown that the HMWS formation rate increased with increasing concentrations of citric acid, especially at concentrations of 10 mM and above. It was shown that the HMWS formation rate increased only slightly with the use of 1,2-propanediol as an uncharged osmolality modifier instead of sucrose. The addition of a neutral amino acid (proline) to the formulations containing sucrose resulted in the lowest HMWS formation rate, even when the sucrose concentration was reduced to 150 mM.

[0149] Table 12: Stability of abatacept (4.25 mg / ml) at 40° C. in formulations 7-1 through 7-18 adjusted to pH 6.8 as assessed by SEC. [Table 12]

[0150] Comparative Example: Effect of Buffer Concentration, Osmotic Adjustment Charge, and Neutral Amino Acids on the Stability of Immunoglobulin G1 (IgG1) at 8-30°C The effect of buffer concentration and osmotic modifier charge on the stability of IgG1 (100 mg / ml) was investigated. Citrate buffer was tested. Sodium chloride (150 mM) was used as the charged osmotic modifier and glycerol (300 mM) was used as the uncharged osmotic modifier. The effect of proline and glycine (50 mM) on IgG1 stability was also investigated in the presence of 1 mM buffer and both osmotic modifiers. All formulations tested contained polysorbate 80 (0.2 mg / ml) and were adjusted to pH 6.0. Table 13 summarizes the formulations tested. All formulations were stressed at 30° C. for 8 weeks. The stability of IgG1 was followed by monitoring the high molecular weight species generation rate using SEC.

[0151] Table 13: Formulations of IgG1 tested. All formulations contained IgG1 (100 mg / ml) and polysorbate 80 (0.2 mg / ml) and were adjusted to pH 6.0. [Table 13]

[0152] All formulations tested passed visual inspection after storage at 30° C. (visual score A). The HMWS formation rate in formulations 8-1 to 8-8 after storage at 30° C. is shown in Table 14. The HMWS formation rate decreased with increasing buffer concentration, both in the presence of sodium chloride and in the presence of glycerol (compare formulations 8-1 to 8-3, and compare formulations 8-4 to 8-6). At low citric acid concentrations (1 or 5 mM), there was a slight trend toward higher ionic strength formulations exhibiting greater stability (compare formulation 8-1 to 8-4, and compare formulation 8-2 to 8-5). At high citric acid concentrations (20 mM), this order was reversed (compare formulation 8-3 to 8-6), although in this example the ionic strength of both formulations was above 70 mM. The presence of neutral amino acids (proline or glycine) resulted in only a slight increase in the HMWS formation rate.

[0153] Table 14: Stability of IgG1 (100 mg / ml) at 30° C. in Formulations 8-1 to 8-8 as assessed by SEC. [Table 14]

[0154] (Summary of the Example) The data of Examples 2-7 show that formulations of engineered protein constructs containing Fc domains are stable with up to 5 mM buffer and low ionic strength (e.g., less than 20 mM excluding the contribution of the engineered protein construct). Higher buffer concentrations, especially above 10 mM, and higher ionic strength, especially above 20 mM (excluding the contribution of the engineered protein construct), were destabilizing. The presence of neutral amino acids was even more stabilizing. Surprisingly, this is the opposite of the behavior exhibited by the four-chain antibody (IgG1 type) tested. The data of Comparative Example 8 show that this antibody was more stable at higher buffer concentrations and higher ionic strength. The presence of neutral amino acids was even more destabilizing.

[0155] Without wishing to be bound by theory, the inventors believe that, compared to native or recombinant proteins based on native structural templates (e.g., immunoglobulins), expression of novel engineered protein constructs containing Fc domains leads to an increased abundance of hydrophobic patches on the protein surface and increased exposure of unstable sites, such as regions susceptible to hydrolytic cleavage. This in turn leads to an increased propensity for aggregation and structural degradation. Without wishing to be bound by theory, the present invention combines formulation features that are believed to act in concert to hide non-native hydrophobic patches and minimize the rate of proton exchange at unnaturally exposed unstable sites, resulting in a substantial increase in the stability of engineered protein constructs containing Fc domains.

[0156] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprises" and "comprising", are understood to mean the inclusion of a stated integer, step, group of integers, or group of steps, but not to the exclusion of any other integer, step, group of integers, or group of steps.

[0157] All patents, patent applications, and literature references mentioned throughout this specification are hereby incorporated by reference in their entirety.

[0158] The present invention includes all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of the above groups.

Claims

1. 1. An aqueous composition having a pH in the range of about 4.0 to about 8.5, comprising: engineered protein constructs containing an Fc domain; Optionally, pK a is in the range of about 3.0 to about 9.5, and a one or more buffering agents, which are substances having at least one ionizable group that is within 2 pH units of the pH of the composition; one or more neutral amino acids, wherein a total concentration of the one or more neutral amino acids in the composition is 20 to 200 mM, and the one or more neutral amino acids comprise neutral amino acids selected from glycine, methionine, proline, and alanine; and Uncharged osmolality regulator Includes; the buffering agent is present in the composition at a total concentration ranging from about 0 mM to about 10 mM; and The aqueous solution composition, wherein the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 20 mM.

2. 2. The aqueous solution composition of claim 1, wherein the buffering agent is present in a total concentration ranging from about 0.1 mM to about 5 mM, such as from about 0.1 mM to about 4 mM, from about 0.1 mM to about 3 mM, or from about 0.1 mM to about 2 mM, or from about 0.1 mM to about 1 mM.

3. 2. The aqueous solution composition of claim 1, wherein the buffering agent is present at a total concentration in the range of about 1 mM to about 5 mM, such as about 1 mM to about 4 mM or about 1 mM to about 3 mM.

4. The buffer has a pK a The aqueous solution composition of any one of claims 1 to 3, comprising an ionizable group that is within 1 unit of the pH of the composition.

5. The buffer(s) may be selected from the group consisting of citric acid, histidine, maleic acid, sulfurous acid, glyoxylic acid, aspartame, glucuronic acid, aspartic acid, glutamic acid, tartaric acid, gluconic acid, lactic acid, glycolic acid, adenine, succinic acid, ascorbic acid, benzoic acid, phenylacetic acid, gallic acid, cytosine, p-aminobenzoic acid, sorbic acid, acetic acid, propionic acid, alginic acid, uric acid, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfone 4. The aqueous solution composition of claim 1, wherein the amine is selected from the group consisting of an acid, piperazine, N,N'-bis(2-ethanesulfonic acid), phosphoric acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof.

6. 6. The aqueous solution composition of claim 5, wherein the buffer is selected from the group consisting of citric acid, histidine, maleic acid, tartaric acid, lactic acid, benzoic acid, acetic acid, bicarbonate, phosphoric acid, and tris(hydroxymethyl)aminomethane, for example, selected from citric acid and phosphoric acid.

7. 7. The aqueous solution composition of claim 1, wherein the osmolality of the composition is in the range of about 200 mOsm / L to about 600 mOsm / L, e.g., about 200 mOsm / L to about 500 mOsm / L, about 200 mOsm / L to about 400 mOsm / L, or about 300 mOsm / L.

8. The aqueous solution composition according to any one of claims 1 to 7, comprising a polyol as an uncharged osmolality adjusting agent.

9. 8. The aqueous solution composition according to claim 1, comprising an uncharged osmotic agent selected from glycerol, 1,2-propanediol, mannitol, sorbitol, sucrose, trehalose, PEG300 and PEG400, in particular selected from glycerol, mannitol, 1,2-propanediol and sucrose.

10. 10. The aqueous solution composition of claim 1, wherein the total concentration of the uncharged osmotic agent is 50 to 1000 mM, for example 200 to 500 mM, or about 300 mM.

11. The aqueous solution composition according to any one of claims 1 to 10, comprising proline as a neutral amino acid.

12. The aqueous solution composition according to any one of claims 1 to 10, comprising glycine as a neutral amino acid.

13. The aqueous solution composition according to any one of claims 1 to 12, wherein a total concentration of the one or more neutral amino acids in the composition is 50 to 150 mM.

14. 14. The aqueous solution composition of any one of claims 1 to 13, wherein the total ionic strength of the composition, excluding the contribution of the engineered protein construct, is less than 10 mM.

15. 15. The aqueous solution composition according to any one of claims 1 to 14, wherein the pH is in the range of about 4.0 to about 7.5, such as about 5.0 to about 7.5, such as about 5.5 to about 7.5, such as about 6.0 to about 7.5, such as about 7.0 to about 7.

5.

16. 16. The aqueous solution composition of any one of claims 1 to 15, wherein the engineered protein construct is a fusion of an Fc domain with a heterologous polypeptide.

17. 17. The aqueous solution composition of claim 16, wherein the heterologous polypeptide is selected from a cytokine, a growth factor, a clotting factor, an enzyme, a receptor protein, a GLP-1 agonist, and functional fragments and domains thereof.

18. 18. The aqueous solution composition of claim 17, wherein the engineered protein construct is selected from etanercept, abatacept, belatacept, aflibercept, rilonacept, romiplostim, eloctate, luspatercept, dulaglutide, and alprolix.

19. 20. The aqueous solution composition of claim 17, wherein the heterologous polypeptide is a protease inhibitor.

20. the engineered protein construct is a bispecific antibody in the format of a four-chain antibody having two different variable binding regions; or 16. The aqueous solution composition of any one of claims 1 to 15, wherein said engineered protein construct is a bispecific antibody in the format of a two-chain antibody having two different variable binding regions.

21. The aqueous solution composition according to any one of claims 1 to 20, wherein the Fc domain is an IgG, for example an IgG1 Fc domain.

22. 22. The aqueous solution composition of any one of claims 1 to 15 or 21, wherein the engineered protein construct comprises an IgG Fc domain and two or more immunoglobulin chain variable domains.

23. the engineered protein construct comprises an IgG Fc domain formed of two chains, each directly linked to one or more (e.g., one or two) immunoglobulin chain variable domains; or 23. The aqueous solution composition of claim 22, wherein the engineered protein construct comprises an IgG Fc domain formed of two chains, each indirectly linked to one or more (e.g., one or two) immunoglobulin chain variable domains.

24. each of said immunoglobulin chain variable domains has the same specificity, or 24. The aqueous solution composition of claim 22 or 23, wherein the construct comprises immunoglobulin chain variable domains with two or more (e.g., two) different specificities.

25. The immunoglobulin chain variable domain is HH Is it a domain? The immunoglobulin chain variable domain is H It is a domain, or The immunoglobulin chain variable domain is L The aqueous solution composition according to any one of claims 22 to 24, which is a domain.

26. 26. The aqueous solution composition of any one of claims 1 to 25, wherein the protein is present in a concentration of 1 to 400 mg / ml, such as 10 to 200 mg / ml, for example 20 to 100 mg / ml.

27. 27. The aqueous solution composition of any one of claims 1 to 26, comprising a non-ionic surfactant selected from the group consisting of alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol, and alkyl phenyl ethers of polyethylene glycol, wherein the non-ionic surfactant is present in a concentration of 10 to 2000 μg / ml, e.g., 50 to 1000 μg / ml, 100 to 500 μg / ml, or about 200 μg / ml.

28. 28. The aqueous solution composition of claim 27, wherein the non-ionic surfactant is a polysorbate, such as polysorbate 20 or polysorbate 80.

29. a preservative, e.g., a phenolic or benzyl preservative selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, and methylparaben; 29. The aqueous solution composition of any one of claims 1 to 28, wherein the preservative is present in a concentration of 10 to 100 mM, such as 20 to 80 mM or 25 to 50 mM.

30. 30. The aqueous solution composition of any one of claims 1 to 29, which is a composition for use in therapy.