Surfactant Stabilizer

Abstract

The present invention relates to stabilized protein-containing formulations, stabilized or inhibited against protein aggregation, comprising an amphiphilic surfactant.

Description

[Technical field]

[0001] The present invention relates to an improved protein-containing formulation stabilized or inhibited against protein aggregation, comprising an amphiphilic surfactant. The formulation comprises a protein, such as a disordered protein, an antibody or an enzyme, and an amphiphilic surfactant as a stabilizer, in particular a vitamin E analogue, a sitosterol analogue or a poly(alkylene glycol) alkyl ether analogue. The use of an amphiphilic surfactant results in an improved formulation that prevents or inhibits protein aggregation. Thus, the present invention provides a stabilized formulation comprising a protein and an amphiphilic surfactant, as well as a method of stabilizing a protein-containing formulation using said surfactant. The present invention further provides the use of an amphiphilic surfactant as a stabilizer for a protein-containing formulation. [Background technology]

[0002] Protein-based pharmaceuticals have a limited shelf life and are often structurally and functionally unstable, so they must be manufactured, transported, and stored using a system of refrigerators and freezers known as the "cold chain," making many of these life-saving drugs difficult and expensive to manufacture and deliver.

[0003] Many molecules are used as crowding agents to stabilize pharmaceuticals in liquid formulations, but these additives can have deficiencies. For example, non-reducing sugars such as mannitol, sorbitol, and trehalose are effective in solution but are prone to crystallization and phase separation upon freezing. (Shire, SJ Curr. Opin. Biotechnol. 20, 708-714 (2009)). Sucrose does not have this problem, but its hydrolysis leads to undesirable glycosylation of pharmaceuticals (Shire, SJ Curr. Opin. Biotechnol. 20, 708-714 (2009)). Surfactants are also common excipients; however, surfactants such as polysorbate 20 and 80 undergo a variety of chemical and enzymatic degradation, and furthermore, they generate peroxides that oxidize methionine groups (Shire, SJ Curr. Opin. Biotechnol. 20, 708-714 (2009)).

[0004] Although some protein-based pharmaceuticals can be stored at room temperature if they are lyophilized (freeze-dried); most protein-based pharmaceuticals denature as a result of either the freezing or drying process. In some cases, surfactants, in addition to crowding agents, can protect protein-based pharmaceuticals during lyophilization, but these crowding agents do not work universally. The most effective additive for a given pharmaceutical depends largely on factors including the drug's isoelectric point, beta-sheet content, and melting temperature (Roughton et al. Comput. Chem. Eng. 58, 369-377 (2013)). Even with the addition of stabilizers, many protein-based pharmaceuticals are too unstable to survive lyophilization (Roughton et al. Comput. Chem. Eng. 58, 369-377 (2013)).

[0005] Surfactants are amphiphilic molecules that are often added to biopharmaceutical formulations to stabilize biological agents (APIs) against stresses encountered during production, transportation, storage and administration. Although the exact mechanism of action depends on the nature of the surfactant and the structural features of the biopharmaceutical to be stabilized, two main mechanisms have been proposed to explain their protective effect: i) the displacement mechanism and ii) selective binding.

[0006] On the negative side, many commercially established surfactants, i.e., polysorbate 20 and polysorbate 80, suffer from various chemical and enzymatic degradation mechanisms, such as autoxidation, hydrolysis, and enzymatic degradation by esterases and lipases. The resulting insoluble free fatty acids can result in visible and non-visible particles, which are of particular concern to regulatory agencies and pose immunogenicity risks. Furthermore, polysorbate degradation products, when exposed to light, can result in the formation of peroxides, which in turn triggers protein oxidation and surfactant autoxidation. Furthermore, polysorbate synthesis can result in impurities or lot-to-lot inconsistencies related to the production of residual free fatty acids, etc., which is another important factor that determines polysorbate stability.

[0007] When preparing a pharmaceutical formulation, which should be physicochemically acceptable and stable over time, not only the physiological properties of the protein must be taken into account, but also other aspects such as industrial production, easy handling for the patient and patient safety. The outcome of these aspects is unpredictable when testing different formulations, and often there is a unique set of solutions for each protein.

[0008] Thus, there is a need in the art to provide stable protein-containing formulations suitable for parenteral administration to a patient, for example, intravenous, intramuscular or subcutaneous administration.

[0009] The present invention overcomes previous deficiencies in the art by providing novel formulations and methods for stabilizing proteins. Summary of the Invention

[0010] The present invention is based on the discovery that significantly higher stabilizing efficacy is achieved by using amphiphilic surfactants as stabilizers in protein-containing formulations compared to conventional stabilizers for pharmaceutical formulations, such as polysorbates.

[0011] Surprisingly, it has been found that amphiphilic surfactants not derived from fatty acid building blocks, such as polysorbates, produce fewer impurities and exhibit a better surfactant stability profile compared to currently used polysorbates.

[0012] Specifically, stable protein-containing formulations with much lower levels of protein aggregation can be prepared by adding amphiphilic surfactants as stabilizers.

[0013] Surprisingly, it was observed that variation in the surfactant linker building blocks significantly affected the surfactant safety profile in primary human tissues, which, to the inventors' knowledge, is unknown in the art.

[0014] Thus, the present invention relates to stable protein-containing formulations comprising amphiphilic surfactants and methods for inhibiting protein aggregate formation by adding amphiphilic surfactants (e.g., vitamin E, sitosterol, poly(alkylene glycol) alkyl ether analogs) as stabilizers to protein-containing formulations.

[0015] In a first aspect, the present invention provides a stable protein-containing formulation comprising an amphiphilic surfactant, wherein the amphiphilic surfactant is not derived from a fatty acid building block.

[0016] In a second aspect, the present invention provides the use of an amphiphilic surfactant as a stabiliser for a formulation comprising a protein.

[0017] In a third aspect, the present invention provides a method for preventing or inhibiting protein aggregate formation in a protein-containing formulation by using an amphiphilic surfactant as a stabilizer in the formulation.

[0018] In a fourth aspect, the present invention provides a compound of formula (I): [ka] or Formula (II): [ka] or Formula (III): [ka] (Relating to formula (I), wherein: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (II), in which: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (III), in which: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; n is 12 to 100, for example, 12 to 80. The present invention provides a compound of the formula:

[0019] Further aspects of the invention are described herein and in the enumerated embodiments.

[0020] The above aspects may be combined. Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and the accompanying embodiments. However, it should be understood that the following description, the accompanying embodiments, and specific examples illustrating preferred embodiments of the present application are given by way of illustration only. Various modifications and changes within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following. [Brief description of the drawings]

[0021] [Figure 1A] Figure 1A shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) surfactant (TPGS1000) in buffer and a combination c) protein and surfactant in buffer. Although the surfactant protein reduces the surface tension throughout the experiment, the surfactant used exceeds the protein surfactant, suggesting that the protein has been displaced from the interface. [Figure 1B] Figure IB shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) surfactant vitamin E dimethyl succinate ((VEDS), Example 10) in buffer and a combination c) protein and surfactant in buffer. Although the surfactant protein reduces the surface tension throughout the experiment, the surfactant used exceeds the protein surfactant, suggesting that the protein has been displaced from the interface. [Figure 1C] Figure 1C shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) surfactant vitamin E dimethyl glutarate (VEDG2.2, Example 12) in buffer and combination c) protein and surfactant in buffer. Although the surfactant protein reduces the surface tension throughout the experiment, the surfactant used exceeds the protein surfactant, suggesting that the protein is displaced from the interface. [Figure 1D] Figure ID shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) surfactant (SPGS-550-M) in buffer and a combination c) protein and surfactant in buffer. Although the surfactant protein reduces the surface tension throughout the experiment, the surfactant used exceeds the protein surfactant, suggesting that the protein has been displaced from the interface. [Figure 1E] Figure 1E shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) detergent (Brij58) in buffer and a combination c) protein and detergent in buffer. Although the detergent used exceeds the protein surface activity throughout the experiment, this suggests that the protein has been displaced from the interface. [Figure 1F] Figure IF shows the equilibrium surface tension of the histidine formulation after 1 hour. The samples measured were a) protein in buffer; b) detergent (Brij58 succinate, Example 1) in buffer and a combination c) protein and detergent in buffer. Although the surfactant protein reduces the surface tension throughout the experiment, the surfactant used exceeds the protein surfactant, suggesting that the protein has been displaced from the interface. [Figure 2A]FIG. 2A shows particle numbers >2 μm / mL generated by orbital shaking of Protein 1 in a liquid formulation for 7 and 24 hours. The assay mimics mechanical stress. The data show less particle generation in disordered protein samples containing amphiphilic surfactants and non-fatty acid based surfactants that outperform the current standard polysorbate. [Figure 2B] FIG. 2B shows particle numbers > 2 μm / mL generated by orbital shaking of protein 2 in a liquid formulation for 7 and 24 hours. The assay mimics mechanical stress. The data show less particle generation in Fab protein samples containing amphiphilic surfactants and non-fatty acid based surfactants that outperform the current standard polysorbate. [Figure 2C] FIG. 2C shows particle numbers >2 μm / mL generated by orbital shaking of protein 3 in a liquid formulation for 7 and 24 hours. The assay mimics mechanical stress. The data show comparable or less particle generation in mAb protein samples containing amphipathic surfactants and non-fatty acid based surfactants over the current standard polysorbate. [Figure 2D] FIG. 2D shows particle numbers > 2 μm / mL generated by orbital shaking of Protein 1 in a liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data show less particle generation in disordered protein samples containing VEDS or VEDG compared to TPGS-1000. [Figure 2E] FIG. 2E shows particle numbers > 2 μm / mL generated by orbital shaking of protein 2 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in Fab protein samples containing vitamin E-based surfactant compared to no surfactant control samples. [Figure 2F]FIG. 2F shows particle numbers > 2 μm / mL generated by orbital shaking of protein 3 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in mAb protein samples containing vitamin E-based surfactant compared to no surfactant control samples. [Figure 2G] FIG. 2G shows particle numbers >2 μm / mL generated by orbital shaking of protein 1 in a liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows particle generation in disordered protein samples containing β-sitosterol-based surfactants compared to no surfactant control samples. [Figure 2H] FIG. 2H shows particle numbers > 2 μm / mL generated by orbital shaking of protein 2 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in Fab protein samples containing β-sitosterol based surfactant compared to no surfactant control samples. [Figure 2I] FIG. 2I shows particle numbers > 2 μm / mL generated by orbital shaking of protein 3 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in mAb protein samples containing β-sitosterol based surfactant compared to no surfactant control samples. [Figure 2J] FIG. 2J shows particle numbers > 2 μm / mL generated by orbital shaking of Protein 1 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in disordered protein samples containing Brij58_succinate compared to protein samples containing Brij58. [Figure 2K]FIG. 2K shows particle numbers > 2 μm / mL generated by orbital shaking of protein 2 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in Fab protein samples containing Brij58-based surfactant compared to no surfactant control samples. [Figure 2L] FIG. 2L shows particle numbers > 2 μm / mL generated by orbital shaking of protein 3 in liquid formulation for 0, 7 and 24 hours. The assay mimics mechanical stress. The data shows less particle generation in mAb protein samples containing Brij58-based surfactant compared to no surfactant control samples. [Figure 3A] Figure 3A shows the % polysorbate content (w / v) after oxidative stress (H2O2) for 6 days at 40°C compared to untreated controls. The data show a slight decrease in PS80 upon H2O2 treatment. [Figure 3B] Figure 3B shows the % polysorbate content (w / v) after pH and temperature stress for 1 month at 50°C compared to the untreated control (FZ). Testing was performed using four representative buffers typically used for biopharmaceuticals. PS80 appears to be stable only in citrate buffer. PS80 is degraded in histidine, phosphate and succinate buffers. [Figure 3C] Figure 3C shows the % (w / v) TPGS1000 content after oxidative stress (H2O2) for 6 days at 40°C compared to untreated controls. TPGS1000 appears to be stable upon H2O2 treatment. [Figure 3D] Figure 3D shows the % TPGS1000 content (w / v) after pH and temperature stress for 1 month at 50°C compared to the untreated control (FZ). The study used four representative buffers used in biopharmaceuticals. TPGS1000 appears to be stable in all buffer conditions used. [Figure 3E]Figure 3E shows the % Brij58 content (w / v) after oxidative stress (H2O2) for 6 days at 40°C compared to untreated controls. Brij58 appears to be stable upon H2O2 treatment. [Figure 3F] Figure 3F shows the % Brij58 content (w / v) after pH and temperature stress for 1 month at 50°C compared to the untreated control (FZ). Testing was performed using four representative buffers used in biopharmaceuticals. Brij58 appears to be stable in citrate and phosphate buffer conditions. Brij58 is degraded in histidine and succinate buffer conditions. [Figure 4A] Figure 4A shows the hemolytic potential of vitamin E-based surfactants compared to polysorbate and no surfactant (NaCl) control samples. The safety profile of the tested vitamin E-based surfactants is comparable to or better than the polysorbate control. [Figure 4B] Figure 4B shows the number (%) of activated B cells upon detergent treatment. Vitamin E derivatives were compared to polysorbate and a B cell activation control (positive). TPGS1000 shows a similar level of B cell activation as polysorbate. Vitamin E derivatives VEDS, VEDG 2.2 and VEDG 3.3 show a lower level of B cell activation than TPGS1000. [Figure 4C] Figure 4C shows the mean fluorescence intensity (MFI) fold change of LPS-activated dendritic cells (DCs) compared to untreated controls and vitamin E derivative-treated cells. The vitamin E derivatives VEDS, VEDG 2.2 and VEDG 3.3 show no DC activation. [Figure 4D] Figure 4D shows the hemolytic potential of beta-sitosterol-based surfactants at increasing concentrations compared to polysorbate and no surfactant (NaCl) control samples. The safety profile of the tested vitamin E-based surfactants is comparable to the polysorbate control. [Figure 4E]Figure 4E shows the number (%) of activated B cells upon detergent treatment. β-sitosterol-based SPGS-550-M was compared to polysorbate and B cell activation control (positive). β-sitosterol shows a slightly increased level of B cell activation that is still considered safe. [Figure 4F] Figure 4F shows the mean fluorescence intensity (MFI) fold change of LPS-activated dendritic cells (DCs) compared to untreated control (cell media) and β-sitosterol-based SPGS-550-M treated cells. SPGS-550-M does not show DC activation. [Figure 4G] FIG. 4G shows the hemolytic potential of Brij58-based surfactants at increasing concentrations compared to polysorbate and no surfactant (NaCl) control samples. Elevated levels of Brij58 (0.05% w / v) show increased hemolytic potential and may be considered unsafe. For Brij58 succinate, which contains a succinate linker between the lipophilic and hydrophilic moieties, the hemolytic potential was reduced. The safety profile of the tested Brij58 succinates is comparable to the polysorbate control. [Figure 4H] Figure 4H shows the number (%) of activated B cells upon detergent treatment. Brij58 and Brij58 succinate treated cells were compared to polysorbate and B cell activation control (positive). Brij58 showed little patient specific B cell activation, which is considered safe on average across all patients. Brij58 succinate showed no B cell activation. [Figure 4I] FIG. 4I shows the mean fluorescence intensity (MFI) fold change of LPS-activated dendritic cells (DCs) compared to untreated control (cell medium), Brij58 succinate and Brij58-treated cells. Brij58 shows little B cell activation at elevated concentrations. Brij58 shows no B cell activation at 0.01% (w / v), the concentration used in biologics. The same is true for Brij58 succinate. [Figure 5A]Figure 5A shows the platelet aggregation inhibition trends of PS80, VEDS and VEDG 3.3. Platelets were activated with four independent agonists. Aggregation was measured by whole blood aggregometry (WBA). VEDS and VEDG 3.3 show similar platelet inhibition values ​​as PS80. [Figure 5B] Figure 5B shows the tendency of PS80 and SPGS-550-M to inhibit platelet aggregation, similar to Figure 5A. Comparison with the negative control shows that SPGS-550-M does not affect platelet aggregation. [Figure 5C] Figure 5C shows the trend of platelet aggregation inhibition of PS80, Brij58 and Brij58-succinate, similar to Figures 5A and 5B. Brij58-succinate has lower inhibition (3 / 4 agonist) than Brij58 on average, which is closer to the level of PS80 and is considered safe. [Figure 6] Figure 6 shows the tendency of detergents to enzymatic degradation resulting from host cell protein (HCP) of protein 4. Detergents tested: PS80, VEDS, VEDG 2.2 and TPGS-1000. PS80 is highly affected. In contrast, VEDS, VEDG 2.2 and TPGS-1000 are not affected by enzymatic degradation after one month, regardless of incubation temperature. [Figure 7] Figure 7 shows the tendency of surfactants towards oxidation and hydrolysis in static stability studies. In the stability studies, formulations of protein 2 containing any of the following surfactants were used: PS80, VEDS, VEDG 2.2 and TPGS-1000, Brij58 and SPGS-550-M. Figure 7 shows that chemical degradation is highly affected for PS80. In contrast, VEDS, TPGS-1000 and Brij58 are not susceptible to degradation to the same extent. SPGS-550-M is susceptible to approximately linear degradation. [Figure 8A]Figure 8A shows the protein purity of formulations containing protein 5. The formulations contained one of the following surfactants: PLX188, PS20, PS80 and VEDS. Formulations containing PLX188, PS80 or VEDS retain similar levels of purity. The stabilizing effect of PS20 is less, i.e., lower purity is observed. [Figure 8B] Figure 8B shows the impurities of the same formulations with protein 5. Formulations containing PLX188, PS80 or VEDS retain similar levels of impurities. The impurity level of PS20 is higher, which complements the purity data from Figure 8A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Surfactants are amphiphilic molecules that are often added to biopharmaceutical formulations to stabilize biopharmaceuticals (APIs) from stresses experienced during manufacture, transportation, storage, and administration. Although the exact mechanism of action depends on the nature of the surfactant and the structural characteristics of the stabilized biopharmaceutical, two main mechanisms have been proposed to explain their protective effect: i) a displacement mechanism and ii) selective binding. Without being bound by any particular theory, amphiphilic antioxidant surfactants, when present in protein-containing formulations, are believed to primarily employ a displacement mechanism, where they preferentially reduce protein adsorption and subsequent protein unfolding and / or aggregation / particle formation phenomena at interfaces (air-liquid; liquid-liquid; liquid-solid), thus reducing interfacial stresses that may otherwise adversely affect protein (API) quality attributes.

[0023] It has been unexpectedly found that protein-containing formulations can be stabilized when amphiphilic antioxidant surfactants are added. Thus, the present invention provides protein-containing compositions with excellent stability and methods for stabilizing protein-containing formulations (by inhibiting aggregate formation).

[0024] The present invention also provides a method for stabilizing proteins using amphiphilic antioxidant surfactants. Additionally, the present invention also provides surfactants of formula (I) and formula (II). The present invention further provides the use of a surfactant of formula (I) or formula (II) for stabilizing a composition containing a biological entity.

[0025] The present invention provides stable protein-containing formulations that include amphiphilic antioxidant surfactants.

[0026] definition The term "amphiphilic surfactant", also referred to as surfactant herein, also refers to non-ionic surfactants such as molecules with a hydrophilic head and a hydrophobic tail. Importantly, the amphiphilic surfactants relevant to the present invention are not derived from fatty acid building blocks, e.g., polysorbates. In one embodiment, the amphiphilic surfactant is of a type and amount such that it is capable of stabilizing a protein-containing composition. The amphiphilic molecular structure can be varied as long as it exhibits the desired properties of preventing protein adsorption and subsequent protein unfolding and / or aggregation / particle formation. Thus, in all aspects and embodiments of the present invention, the amphiphilic surfactant is one that is not derived from a fatty acid building block. The general structure of an amphiphilic surfactant is shown below, where R * is a hydrophilic head group, and R ** is the hydrophobic tail. [ka] R * R may be selected from any group in a polar head configuration. * can be selected from highly polar or hydrophilic moieties, as well as less hydrophilic moieties containing alkyl chains. The hydrophilic head of the amphiphilic antioxidant surfactant preferably comprises a poly(C1-C4 alkylene) glycol group, e.g., a PEG group. The amphiphilic surfactant is preferably selected from vitamin E analogues, sitosterol analogues, and polyethylene glycol alkyl ether analogues, e.g., poly(alkylene glycol) alkyl ether analogues.

[0027] The term "amphiphilic antioxidant surfactant" refers to a non-ionic surfactant such as a molecule with a hydrophilic head and a hydrophobic tail that has antioxidant or radical scavenging properties. In one embodiment, the amphiphilic antioxidant is of a type and in an amount that is capable of stabilizing a protein-containing composition. The amphiphilic antioxidant molecular structure can vary so long as it has antioxidant or radical scavenging properties and exhibits the desired properties of preventing protein adsorption and subsequent protein unfolding and / or aggregation / particle formation. The general structure of an amphiphilic antioxidant surfactant is shown below, where R * is a hydrophilic head group, and R ** is the hydrophobic tail. [ka] R * R may be selected from any group with a polar head configuration that also has suitable radical scavenging ability. * can be selected from highly polar or hydrophilic moieties as well as less hydrophilic moieties containing alkyl chains, so long as it provides adequate radical scavenging functionality to protect or inhibit attack of the surfactant by oxidizing agents. The hydrophilic head of the amphiphilic antioxidant surfactant preferably comprises a poly(C1-C4 alkylene) glycol group, e.g., a PEG group. The amphiphilic antioxidant surfactant is preferably a vitamin E analogue.

[0028] The term "not derived from fatty acid building blocks" in relation to the amphiphilic surfactants of the present disclosure means that the lipophilic core does not contain a fatty acid moiety, for example, it is not a derivative of sorbitol esterified with a fatty acid, such as polysorbate. The terms "not derived from fatty acid building blocks" and "not derived from fatty acid building blocks" are used interchangeably. In one embodiment, the term "not derived from fatty acid building blocks" means "not polysorbate".

[0029] The term "vitamin E analog" refers to compounds that have a structural motif of the vitamin E family, specifically, a chroman moiety, and can be modified in various ways to improve their stabilizing effect in protein-containing formulations. The phrases "vitamin E analog" and "vitamin E derivative" are used interchangeably.

[0030] The term "sitosterol analog" refers to a compound that has a structural motif of the plant sterol family (e.g., β-sitosterol), specifically, a tetracyclic cyclopenta-α-phenanthrene ring, and can be modified in various ways to improve the stabilizing effect in protein-containing formulations. The phrases "sitosterol analog" and "sitosterol derivative" are used interchangeably.

[0031] The term "poly(alkylene glycol) alkyl ether analog" refers to a poly(alkylene glycol) group having a carbon chain of 12 or more carbon atoms, as shown below, e.g., a PEG group. [ka] where p is at least 1, preferably 1 to 100. Poly(alkylene glycol) alkyl ether analogs include Brij surfactants. Poly(alkylene glycol) alkyl ether analogs that can be used in the present disclosure can be modified in various ways to improve the stabilizing effect in protein-containing formulations.

[0032] For example, the aliphatic linear alcohol moiety can be separated from the terminal poly(alkylene glycol) group via a linking group. The linking group (L) is an aliphatic linear alcohol core structure and a poly(alkylene glycol) group, e.g., [ka] wherein p is at least 1, and preferably 1-100.

[0033] The term "dicarboxylic acid linking group" refers to a linking group X of the formula -C(=O)-XC(=O)-, where the linking group X is derived from a dicarboxylic acid or anhydride precursor and has an ester bond to the core structure and R 1 X represents a linear or branched alkylene chain having at least two carbon atoms. In certain embodiments, at least one carbon atom of the branched alkylene chain of X is bonded to two C1-C4 alkyl, e.g., methyl, substituents. In certain embodiments, the branched alkylene chain of X is bonded to two C2-C4 alkyl, e.g., methyl, substituents. 20 It is alkylene.

[0034] As used herein, "C1-C 10 The term "alkyl" refers to a straight or branched hydrocarbon chain radical containing no unsaturation, having from 1 to 10 carbon atoms, and consisting solely of carbon and hydrogen atoms, attached to the remainder of the molecule by a single bond. 20 Alkyl, C5-C 15 Alkyl, C 10 ~C 15The terms "alkyl", "C1-C6 alkyl", and "C1-C4 alkyl" should be construed accordingly. 10 Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (t-butyl), n-pentyl, n-hexyl, n-heptyl, 4-heptyl, n-octyl, 2-isopropyl-3-methylbutyl, n-nonyl, and n-decyl.

[0035] As used herein, "C2-C 20 The term "alkylene" refers to a straight or branched hydrocarbon chain divalent radical, containing no unsaturation, consisting solely of carbon and hydrogen atoms, having from 2 to 20 carbon atoms. 15 Alkylene, C2-C 10 "C2-C6 alkylene", "C3-C 15 Alkylene, C3-C 10 "C3-C6 alkylene", "C4-C 15 The terms "alkylene" and "C4-C6 alkylene" should be interpreted accordingly. 20 Examples of "alkylene" include, but are not limited to, n-propylene, 2,2-dimethylpropylene, ethylene, 2-methylprop-2-ylene, n-butylene, 1-methylpropylene (sec-butylene), 2-methylpropylene (iso-butylene), 1,1-dimethylethylene (t-butylene), n-pentylene, n-hexylene, n-heptylene, 4-heptylene, n-octylene, 2-isopropyl-3-methylbutylene, n-nonylene, n-decylene, and n-eicosylene.

[0036] The term "protein" as used herein is used according to its conventional meaning, i.e., as a sequence of amino acids. Proteins are not limited to a particular length, e.g., they may include full-length protein sequences (or peptides) or fragments of full-length proteins, and may include non-natural amino acids, e.g., D-amino acids. Proteins may include post-translational modifications of proteins, e.g., glycosylation, acetylation, phosphorylation, and the like, as well as other modifications, both natural and non-natural, known in the art. Proteins useful in the formulations of the present disclosure may be prepared using any of a variety of well-known recombinant and / or synthetic techniques, illustrative examples of which are further described below. The protein of the present invention is preferably a therapeutic protein, and may be any protein-based molecule (e.g., biologic), including, but not limited to, an antibody drug conjugate, a monoclonal antibody (mAb), a fragment antigen-binding fragment (F(ab)) or an enzyme, or an Fc fragment containing one or more fusion proteins.

[0037] The term "protein-containing formulation" as used herein refers to a liquid pharmaceutical composition that contains a therapeutic protein as an active pharmaceutical ingredient (API) formulated together with one or more pharma-ceutically acceptable vehicles. The protein-containing formulation is suitable for patient administration and is used to provide a therapeutic effect to the patient. The protein-containing formulation of the present invention is a solution (also called a protein-containing liquid formulation). In some embodiments, the therapeutic protein is present in a unit dose suitable for administration in a treatment regimen. The protein-containing formulation may include, but is not limited to, a disordered protein, an antibody, or an enzyme.

[0038] The term "stable protein-containing formulation" refers to a formulation comprising an amphiphilic surfactant as described herein. Preferably, the term "stable protein-containing formulation" refers to a formulation in which protein aggregation is avoided or inhibited, in particular in which degradation, such as the formation of insoluble and soluble aggregates, is avoided or inhibited during storage in liquid or frozen conditions.

[0039] The terms "contact," "contacting," and "contacted," and grammatical conjugations thereof, refer to bringing together components of a desired reaction under conditions suitable for carrying out the desired reaction (e.g., stabilization of a protein). The term "contacting" can include any method by which a protein is exposed, provided, or an amphiphilic surfactant is applied.

[0040] As used herein, "stabilization" of a protein-containing formulation means maintaining the structure (1°, 2°, 3° and / or 4° structure) and function of the protein under either aqueous or dry conditions, or after being frozen and / or dried and then thawed and / or rehydrated. In certain embodiments, when a protein is contacted with an amphiphilic surfactant, the protein may be stable at temperatures from about -80°C to about 100°C.

[0041] The term "antibody" as used herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), modified antibodies, antibody drug conjugates, and antibody fragments that exhibit the desired biological activity. The natural form of an antibody is a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to an antigen. The light and heavy chain variable domains consist of framework regions mediated by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The constant regions are recognized by and can interact with the immune system. (See, for example, Janeway et al., 2001, Immunol. Biology, 5th Ed., Garland Publishing, New York). The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antibody can be derived from any suitable species. In certain embodiments, the antibody is of human or non-human, e.g., murine, origin. The antibody can be, for example, human, humanized, or chimeric. The antibodies used in the present invention are not particularly limited, so long as they bind to the antigen of interest.

[0042] The term "modified antibody" as used herein refers to an antibody linked to various molecules such as polyethylene glycol (PEG) or a cytotoxic drug (Farmaco. 1999 Aug 30; 54(8): 497-516; Cancer J. 2008 May-Jun; 14(3): 154-69). The "antibody" used in the present invention also includes such modified antibodies. Such modified antibodies can be prepared by chemically modifying the obtained antibody. Such methods have already been established in the art.

[0043] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0044] An "intact antibody" is one which contains an antigen-binding variable region as appropriate for the antibody class, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3 and CH4. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0045] "Antibody fragments" include portions of an intact antibody, including its antigen-binding or variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0046] An "antigen" is an entity to which an antibody specifically binds.

[0047] The terms "specific binding" and "specifically bind" mean that an antibody or antibody derivative binds with high selectivity to its corresponding epitope of a target antigen and not to many other antigens. Typically, an antibody or antibody derivative binds with a high selectivity of at least about 1×10 -7 M, preferably 10 -8 M~10-9 M, 10 -10 M, 10 -1 1M or 10 -12 M and binds to a given antigen with an affinity that is at least two-fold higher than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0048] The terms "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent completely.

[0049] The term "therapeutically effective amount" refers to an amount of a protein effective to treat a disease or disorder in a mammal.

[0050] The term "antibody drug conjugate" or "ADC" as used herein refers to a compound linked to a cell binding agent (i.e., an antibody or a fragment thereof). Typically, the cell binding agent (e.g., an antibody) is covalently attached to the drug by a linker.

[0051] As used herein, the term "isolated" refers to a protein or peptide that is free or substantially free from other macromolecular species found in a cellular environment. Proteins used in the present invention may be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques readily recognized in the art.

[0052] It should be understood that the term "about" allows for standard deviation, as understood by one of ordinary skill in the art; when ranges are given, the endpoints are included.

[0053] Enumerated Embodiments Embodiment 1. Formula (I): [ka] or Formula (II): [ka] or Formula (III): [ka] (Relating to formula (I), wherein: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (II), in which: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (III), in which: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; n is 12 to 100, for example, 12 to 80. Compound.

[0054] Embodiment 2. Formula (I), (II) or (III) (wherein R 1is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, wherein the poly(C1-C4 alkylene glycol) group terminates with a hydroxyl or a C1-C6 alkoxyl.

[0055] Embodiment 3. Formula (I), (II) or (III) (wherein R 1 But -O-(CH2-CH2) m -R 1a where R 1a is selected from hydroxyl and C1-C6 alkoxyl, and m is an integer from 1 to 80.

[0056] Embodiment 4. The compound of embodiment 3, having formula (I), (II) or (III), wherein m is an integer from 1 to 50.

[0057] Embodiment 5. A compound according to any one of embodiments 3 and 4, having formula (I), (II) or (III), wherein m is an integer from 1 to 30.

[0058] Embodiment 6. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each independently C1 to C6 alkyl) or formula (II) (wherein R 4 and R 5 and each is independently C1-C6 alkyl.

[0059] Embodiment 7. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each independently C1-C6 alkyl; R 6 C5~C 15 alkyl) or formula (II) 4 and R 5 are each independently C1-C6 alkyl; R6 C5~C 15 The compound according to any one of the preceding embodiments, wherein R is an alkyl group.

[0060] Embodiment 8. Formula (I) or (II) (wherein R 6 But branched C 10 ~C 15 The compound according to any one of the preceding embodiments, wherein R is an alkyl group.

[0061] Embodiment 9. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each independently C1-C4 alkyl) or formula (II) (wherein R 4 and R 5 is each independently C1-C4 alkyl.

[0062] Embodiment 10. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each methyl) or formula (II) 4 and R 5 and each is methyl.

[0063] Embodiment 11. A compound of formula (I), (II) or (III) (wherein L 1 is a branched dicarboxylic acid linking group comprising at least one carbon atom bonded to two C1-C4 alkyl substituents, e.g., methyl.

[0064] Embodiment 12. A compound of formula (I), (II) or (III) (wherein L 1 is -C(=O)-XC(=O)-, and X is a branched C2-C 20 Alkylene, C2-C 15 Alkylene or C2-C 1012. The compound according to any one of the preceding embodiments, wherein R is an alkylene.

[0065] Embodiment 13. A compound of formula (I), (II) or (III) (wherein L 1 The compound according to any one of embodiments 1 to 12, wherein:

[0066] Embodiment 14. Formula (I), (II) or (III) (wherein branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 12 and 13, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two C1-C4 alkyl groups.

[0067] Embodiment 15. Formula (I), (II) or (III) (wherein X is a branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 12 to 14, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two methyl substituents.

[0068] Embodiment 16. Formula (I), (II) or (III) (wherein X is a branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10The compound according to any one of embodiments 12 to 15, wherein 1, 2 or 3 carbon atoms of alkylene or C2-C6C alkylene are bonded to two C1-C4 alkyl groups, optionally two methyl substituents.

[0069] Embodiment 17. Formula (I), (II) or (III) (wherein X is a branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 20 Alkylene, C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 12 to 16, wherein one carbon atom of the alkylene or C2-C6C alkylene is bonded to two C1-C4 alkyl groups, optionally two methyl substituents.

[0070] Embodiment 18. A compound of formula (I) wherein: L 1 is -C(=O)-XC(=O)-; X is a branched C2-C 20 is alkylene; R 1 is a poly(ethylene glycol) group (PEG), the poly(ethylene glycol) group being terminated with a hydroxyl or a C1-C6 alkoxyl; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 However, branched C5~C 15 The compound of embodiment 1, wherein R is an alkyl group.

[0071] Embodiment 19. A compound of formula (I) (wherein R 1 But -O-(CH2-CH2) m -R 1a where R 1ais selected from hydroxyl and C1-C6 alkoxyl; and m is an integer from 1 to 80, 1 to 50, or 1 to 30.

[0072] Embodiment 20. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 is each independently C1-C6 alkyl.

[0073] Embodiment 21. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each independently C1-C6 alkyl, optionally methyl; R 6 However, branched C5~C 15 21. The compound according to any one of embodiments 1 and 18-20, wherein R is an alkyl group.

[0074] Embodiment 22. A compound of formula (I) (wherein R 6 But branched C 10 ~C 15 22. The compound according to any one of embodiments 1 and 18-21, wherein R is an alkyl group.

[0075] Embodiment 23. A compound of formula (I) (wherein R 2 , R 3 , R 4 and R 5 are each independently C1-C4 alkyl, and optionally, R 2 , R 3 , R 4 and R 5 and each are methyl.

[0076] Embodiment 24. Formula (I) (wherein X is a branched C2-C 15 Alkylene or branched C3-C 15The compound according to any one of embodiments 1 and 18-23, wherein R is an alkylene.

[0077] Embodiment 25. Formula (I) (wherein X is a branched C2-C 10 Alkylene or branched C3-C 10 25. The compound according to any one of embodiments 1 and 18-24, wherein R is an alkylene.

[0078] Embodiment 26. The compound according to any one of embodiments 1 and 18 to 25, represented by formula (I) (wherein X is a branched C2-C6 alkylene, or a branched C3-C6 alkylene).

[0079] Embodiment 27. Formula (I) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 18 to 26, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two C1-C4 alkyl groups.

[0080] Embodiment 28. Formula (I) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 18-27, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two methyl substituents.

[0081] Embodiment 29. Formula (I) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10The compound according to any one of embodiments 1 and 18-28, wherein 1, 2 or 3 carbon atoms of the alkylene or C2-C6C alkylene are bonded to a C1-C4 alkyl group, optionally two methyl substituents.

[0082] Embodiment 30. A compound according to any one of embodiments 1 and 18-29, having formula (I) where X is 2,2-dimethylpentylene or 3,3-dimethylpentylene.

[0083] Embodiment 31. A compound represented by formula (II) wherein: L 1 is -C(=O)-XC(=O)-; X is a branched C2-C 20 is alkylene; R 1 is a PEG group, the PEG group being terminated with a hydroxyl or a C1-C6 alkoxyl; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 However, branched C5~C 15 The compound of embodiment 1, wherein R is an alkyl group.

[0084] Embodiment 32. A compound represented by formula (II) R 1 But -O-(CH2-CH2) m -R 1a where R 1a is selected from hydroxyl and C1-C6 alkoxyl; and m is an integer from 1 to 80, 1 to 50, or 1 to 30.

[0085] Embodiment 33. A compound of formula (II) (wherein R 4 and R 5 and each is independently C1-C6 alkyl.

[0086] Embodiment 34. A compound of formula (II) (wherein R 4 and R 5 are each independently C1-C6 alkyl, optionally methyl; R 6 However, branched C5~C 15 The compound according to any one of embodiments 1 and 31-33, wherein R is an alkyl group.

[0087] Embodiment 35. A compound of formula (II) (wherein R 6 But branched C 10 ~C 15 The compound according to any one of embodiments 1 and 31-34, wherein R is an alkyl group.

[0088] Embodiment 36. A compound of formula (II) (wherein R 4 and R 5 are each independently C1-C4 alkyl, and optionally, R 4 and R 5 and each is methyl.

[0089] Embodiment 37. Formula (II) (wherein X is a branched C2-C 15 Alkylene or branched C3-C 15 The compound according to any one of embodiments 1 and 31-36, wherein R is an alkylene.

[0090] Embodiment 38. Formula (II) (wherein X is a branched C2-C 10 Alkylene or branched C3-C 10 38. The compound according to any one of embodiments 1 and 31-37, wherein R is an alkylene.

[0091] Embodiment 39. The compound according to any one of embodiments 1 and 31 to 38, represented by formula (II) (wherein X is a branched C2-C6 alkylene or a branched C3-C6 alkylene).

[0092] Embodiment 40. Formula (II) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 31-39, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two C1-C4 alkyl groups.

[0093] Embodiment 41. Formula (II) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 31-40, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two methyl substituents.

[0094] Embodiment 42. Formula (II) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 31-41, wherein 1, 2 or 3 carbon atoms of alkylene or C2-C6C alkylene are bonded to two C1-C4 alkyl groups, optionally two methyl substituents.

[0095] Embodiment 43. A compound according to any one of embodiments 1 and 31-42, having the formula (II) where X is 2,2-dimethylpentylene or 3,3-dimethylpentylene.

[0096] Embodiment 44. A compound represented by formula (III) wherein: L 1 is -C(=O)-XC(=O)-; X is a branched C2-C20 is alkylene; R 1 is a PEG group, the PEG group being terminated with a hydroxyl or a C1-C6 alkoxyl; and n is 12 to 100, for example, 12 to 80.

[0097] Embodiment 45. A compound of formula (III) (wherein R 1 But -O-(CH2-CH2) m -R 1a where R 1a is selected from hydroxyl and C1-C6 alkoxyl, and m is an integer from 1 to 80, 1 to 50, or 1 to 30.

[0098] Embodiment 46. Formula (III) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6 alkylene, or X is a branched C3-C 15 Alkylene, C3-C 10 The compound according to any one of embodiments 1, 44, and 45, wherein R is C1-C6 alkylene or C3-C6 alkylene.

[0099] Embodiment 47. Formula (III) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 44-46, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two C1-C4 alkyl groups.

[0100] Embodiment 48. Formula (III) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 44-47, wherein at least one carbon atom of the alkylene or C2-C6C alkylene is bonded to two methyl substituents.

[0101] Embodiment 49. Formula (III) (wherein X is a branched C2-C 15 Alkylene, C2-C 10 alkylene or C2-C6C alkylene, where branched C2-C 15 Alkylene, C2-C 10 The compound according to any one of embodiments 1 and 44-48, wherein 1, 2 or 3 carbon atoms of alkylene or C2-C6C alkylene are bonded to two C1-C4 alkyl groups, optionally two methyl substituents.

[0102] Embodiment 50. The compound of any one of embodiments 1 and 44-49, having the formula (III) where X is 2,2-dimethylpentylene or 3,3-dimethylpentylene.

[0103] Embodiment 51. The compound according to any one of embodiments 1, 11-17, and 44-50, having formula (III) wherein n is 12-50, and optionally n is 12-30.

[0104] Embodiment 52. Formula (I) is a compound of formula (Ia): [ka] The compound according to any one of embodiments 1 to 30, wherein

[0105] Embodiment 53. Formula (I) is a compound of formula (Ia)-i: [ka] The compound of any one of embodiments 1 to 30 and 52, wherein

[0106] Embodiment 54. Formula (II) is a compound of formula (IIa): [ka] The compound according to any one of embodiments 1 to 17 and 31 to 43,

[0107] Embodiment 55. Formula (II) is a compound of formula (IIa)-i: [ka] The compound of any one of embodiments 1-17, 31-43, and 54, wherein

[0108] Embodiment 56. A protein-containing formulation comprising an amphiphilic surfactant, wherein the amphiphilic surfactant is present in an amount sufficient to improve the stability of the protein against aggregation.

[0109] Embodiment 57. The protein-containing formulation of embodiment 56, wherein the amphiphilic surfactant is an amphiphilic antioxidant surfactant.

[0110] Embodiment 58. The protein-containing formulation of embodiment 56, wherein the amphiphilic surfactant is a vitamin E analogue, a poly(alkylene glycol) alkyl ether analogue, or a sitosterol analogue.

[0111] Embodiment 59. The protein-containing formulation of embodiment 57, wherein the amphiphilic antioxidant surfactant is a vitamin E analogue.

[0112] Embodiment 60. A protein-containing formulation according to any one of embodiments 56 to 59, wherein the amphiphilic surfactant comprises a poly(alkylene glycol) moiety, such as a poly(C1-C4 alkylene glycol) moiety, such as PEG.

[0113] Embodiment 61. The amphiphilic surfactant has the formula (I)-i: [ka] or Formula (II)-i: [ka] or Formula (III): [ka] (In the formula: Regarding formula (I)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (II)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (III): L 1 is a linear or branched dicarboxylic acid linking group; R 1is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; n is 12 to 100, for example, 12 to 80. The protein-containing formulation according to any one of embodiments 56 to 60, wherein the compound is represented by the formula:

[0114] Embodiment 62. A protein-containing formulation described in any one of embodiments 56 to 61, wherein the amphiphilic surfactant is a compound represented by formula (I), (II) or (III) described in any one of embodiments 1 to 55.

[0115] Embodiment 63. A protein-containing formulation according to any one of embodiments 56 to 62, wherein the amphiphilic surfactant is present in an amount 4 to 20 times, optionally 4 to 10 times, optionally 4, 5, 6, 10 or 20 times greater than the critical micelle concentration.

[0116] Embodiment 64. A protein-containing formulation according to any one of embodiments 56 to 63, wherein the amphiphilic surfactant is present in an amount of 0.001 to 0.5% (w / v), optionally 0.01 to 0.1% (w / v).

[0117] Embodiment 65. A protein-containing formulation according to any one of embodiments 56 to 64, wherein the protein is present in an amount of 1 to 250 mg / ml.

[0118] Embodiment 66. A protein-containing formulation according to any one of embodiments 56 to 65, wherein the protein is an antibody.

[0119] Embodiment 67. The protein-containing formulation of embodiment 66, wherein the antibody is an antibody-drug conjugate (ADC), a monoclonal antibody (mAB) or an antigen-binding fragment (Fab).

[0120] Embodiment 68. The protein-containing formulation of embodiment 67, wherein the monoclonal antibody is a humanized or human antibody.

[0121] Embodiment 69. The protein-containing formulation of embodiment 66, wherein the antibody is present in an amount of 1 to 250 mg / ml.

[0122] Embodiment 70. A protein-containing formulation according to any one of embodiments 56 to 69, which is an aqueous formulation.

[0123] Embodiment 71. A protein-containing formulation according to any one of embodiments 56 to 70, for subcutaneous administration.

[0124] Embodiment 72. Use of an amphiphilic surfactant as a stabilizer for a formulation containing a protein.

[0125] Embodiment 73. The use according to embodiment 72, wherein the amphiphilic surfactant is defined according to any one of embodiments 57 to 64.

[0126] Embodiment 74. The use according to any one of embodiments 71 and 72, wherein the protein is defined according to any one of embodiments 65 to 69.

[0127] Embodiment 75. A method for improving the stability of a formulation comprising a protein, comprising incorporating an amphiphilic surfactant into the solution, wherein the amphiphilic surfactant component is present in an amount sufficient to improve the stability of the surfactant against aggregation.

[0128] Embodiment 76. The method of embodiment 75, comprising contacting the protein with at least one amphiphilic surfactant to produce a liquid formulation comprising the protein and the amphiphilic surfactant, thereby stabilizing the protein.

[0129] Embodiment 77. The method according to any one of embodiments 75 and 76, wherein the amphiphilic surfactant is defined according to any one of embodiments 57 to 64.

[0130] Embodiment 78. The method according to any one of embodiments 75 to 77, wherein the protein is defined according to any one of embodiments 65 to 69.

[0131] Embodiment 79. A method for preventing or inhibiting protein aggregate formation in a protein-containing formulation by using an amphiphilic surfactant as a stabilizer in the formulation.

[0132] Embodiment 80. The method according to embodiment 79, wherein the method is for preventing or inhibiting protein aggregate formation during storage at room temperature or during frozen storage of the protein-containing formulation.

[0133] Embodiment 81. The method according to any one of embodiments 79 and 80, wherein the amphiphilic surfactant is defined according to any one of embodiments 57 to 64.

[0134] Embodiment 82. The method according to any one of embodiments 79 to 81, wherein the protein is defined according to any one of embodiments 65 to 69.

[0135] Embodiment 83. A method for preparing a stable protein-containing formulation, comprising contacting the protein with at least one amphiphilic surfactant to produce a liquid formulation comprising the protein and the amphiphilic surfactant, wherein the amphiphilic surfactant component is added in an amount sufficient to improve the stability of the surfactant against aggregation.

[0136] Embodiment 84. The method of embodiment 83, wherein the amphiphilic surfactant is defined according to any one of embodiments 57 to 64.

[0137] Embodiment 85. The method according to any one of embodiments 83 and 84, wherein the protein is defined according to any one of embodiments 65 to 69.

[0138] Embodiment 86. A stable protein-containing formulation comprising an amphiphilic surfactant.

[0139] Embodiment 87. A protein-containing formulation according to embodiment 86, wherein the amphiphilic surfactant is defined according to any one of embodiments 57 to 64.

[0140] Embodiment 88. A protein-containing formulation according to any one of embodiments 86 and 87, wherein the protein is defined according to any one of embodiments 65 to 69.

[0141] Embodiment 89. A protein-containing formulation according to any one of embodiments 86 to 88, wherein the formulation is defined according to any one of embodiments 70 and 71.

[0142] Embodiment 1a. A stable protein-containing formulation comprising an amphiphilic surfactant, wherein the amphiphilic surfactant is not derived from a fatty acid building block.

[0143] Embodiment 2a. The formulation of embodiment 1a, wherein the amphiphilic surfactant is an amphiphilic antioxidant surfactant.

[0144] Embodiment 3a. A formulation according to embodiment 1a or 2a, wherein the amphiphilic surfactant is a vitamin E analogue.

[0145] Embodiment 4a. The formulation of embodiment 1a, wherein the amphiphilic surfactant is a poly(alkylene glycol) alkyl ether analogue or a sitosterol analogue.

[0146] Embodiment 5a. The formulation of any one of the preceding embodiments 1a-4a, wherein the protein is a disordered protein or an antibody.

[0147] Embodiment 6a. The formulation of embodiment 5a, wherein the antibody is an antibody-drug conjugate, a monoclonal antibody or an antigen-binding fragment.

[0148] Embodiment 7a. The formulation of any one of embodiments 5a and 6a, wherein the antibody is present in an amount of 1 to 250 mg / ml.

[0149] Embodiment 8a. A formulation according to any one of embodiments 3a-7a, wherein the vitamin E analog, poly(alkylene glycol) alkyl ether analog or sitosterol analog comprises a poly(alkylene glycol) moiety as the hydrophilic portion of the surfactant molecule.

[0150] Embodiment 9a. The amphiphilic surfactant has formula (I)-i: [ka] or Formula (II)-i: [ka] or Formula (III): [ka] (In the formula: Regarding formula (I)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (II)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (III): L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; The formulation of any one of the preceding embodiments 1a-8a, wherein n is 12 to 100, e.g., 12 to 80.

[0151] Embodiment 10a. The amphiphilic surfactant is represented by formula (I) or (II) or (III) L 1 is -C(=O)-XC(=O)-; X is a branched C2-C 20 is alkylene; R 1 is a PEG group, the PEG group being terminated with a hydroxyl or a C1-C6 alkoxyl; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 The formulation of embodiment 9a, wherein the compound is represented by the formula:

[0152] Embodiment 11a. A formulation according to any one of embodiments 1a to 10a, which is for subcutaneous administration.

[0153] Embodiment 12a. Use of an amphiphilic surfactant as a stabilizer for a formulation comprising a protein, wherein the amphiphilic surfactant is not derived from a fatty acid building block.

[0154] Embodiment 13a. The use according to embodiment 12a, wherein the amphiphilic surfactant is defined according to any one of embodiments 1a to 4a, and 8a to 10a.

[0155] Embodiment 14a. The use according to any one of embodiments 12a and 13a, wherein the protein is defined according to any one of embodiments 5a to 7a.

[0156] Embodiment 15a. A method for preventing or inhibiting protein aggregate formation in a protein-containing formulation by using an amphiphilic surfactant not derived from a fatty acid building block as a stabilizer in the formulation.

[0157] Embodiment 16a. The method of embodiment 15a, wherein the amphiphilic surfactant is defined according to any one of embodiments 1a to 4a, and 8a to 10a.

[0158] Embodiment 17a. The method according to any one of embodiments 15a and 16a, wherein the protein is defined according to any one of embodiments 5a to 7a.

[0159] Embodiment 18a. Formula (I): [ka] or Formula (II): [ka] or Formula (III): [ka] (Relating to formula (I), wherein: L 1 is a branched dicarboxylic acid linking group; R1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (II), in which: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (III), in which: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; n is 12 to 100, for example, 12 to 80. Surfactant.

[0160] Surfactants of formula (I) or (II) or (III) The present invention relates to a compound of formula (I): [ka] or Formula (II): [ka] or Formula (III): [ka] (Relating to formula (I), wherein: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 2 , R 3 , R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (II), in which: L 1 is a branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; With respect to formula (III), in which: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, for example, a PEG group; n is 12 to 100, for example, 12 to 80. The present invention provides a surfactant comprising:

[0161] The surfactant compounds of formula (I) or (II) or (III) are 1The poly(C1-C4 alkylene glycol) group may have an average molecular weight in the range of about 100 to about 10,000 g / mol, particularly in the range of about 200 to about 5,000 g / mol, particularly in the range of about 250 to 2500 g / mol, about 400 to about 2,000 g / mol, or about 500 to 1500 g / mol, for example, about 550, about 750, or about 1000 g / mol.

[0162] residue R 1 The poly(C1-C4 alkylene glycol) group, for example, poly(ethylene) glycol, poly(propylene) glycol, is 1 The linking group can be a branched dicarboxylic acid linking group for formula (I) and (II) and a linear or branched dicarboxylic acid linking group for formula (III). Such linking groups form ester bonds to the core structure and to the poly(C1-C4 alkylene glycol) group. In particular, L 1 The linear dicarboxylic acid linking group of L may be succinic acid, sebacic acid, glutaric acid, adipic acid, maleic acid or fumaric acid. 1 The branched dicarboxylic acid linking groups include at least one carbon atom bonded to two C1-C4 alkyl groups, preferably two methyl substituents. For example, exemplary linking groups including branched dicarboxylic acid derivatives having 2-20 carbon atoms are 2,2-dimethylsuccinic acid, 2,2-dimethylglutaric acid, and 3,3-dimethylglutaric acid.

[0163] It has been found that when at least one carbon atom of the branched dicarboxylic acid linking group is bonded to two C1-C4 alkyl groups, e.g., methyl, the branched dicarboxylic acid linking group allows for better protection of the surfactant against chemical and enzymatic degradation of adjacent ester groups.

[0164] In certain embodiments, R 1The poly(C1-C4 alkylene glycol) group further comprises an end group bonded to the end of the polyethylene glycol group. The end group is in particular bonded to the polyethylene glycol group via an ether, ester or amide bond, preferably the end group is in particular bonded to the polyethylene glycol group via an ether bond. The end group is in particular a hydroxyl or C1-C6 alkoxyl group, in particular a methoxyl (-OCH3) group.

[0165] R 2 , R 3 , R 4 and R 5 are independently hydrogen or C1-C 10 alkyl, in particular hydrogen, methyl or ethyl, in particular hydrogen or methyl. In certain embodiments, R 5 is methyl. In certain embodiments, R 4 is methyl. In certain embodiments, R 4 and R 5 is methyl. In a further embodiment, R 2 and R 3 is independently hydrogen or methyl. In certain embodiments, R 4 and R 5 is methyl and R 2 and R 3 is independently hydrogen or methyl. In certain embodiments where the surfactant has formula (I), R 2 , R 3 , R 4 and R 5 are all methyl. In certain embodiments where the surfactant has formula (II), R 4 and R 5 Both are methyl.

[0166] R 6 But C5~C 20 It is an alkyl group. 6 may be linear or branched and unsubstituted. In particular, R 6 is branched.

[0167] In embodiments where the surfactant is represented by formula (I), R 6 But C5~C 20 Alkyl, especially C 10 ~C 15 Alkyl, especially branched C 10 ~C 15 In certain embodiments, R 6 But branched C 15 alkyl, in particular 4,8,12-trimethyltridecyl. In certain embodiments, R 5 and R 6 The carbon atom bonded to has the R configuration.

[0168] In embodiments where the surfactant has formula (II), R 6 But C5~C 20 Alkyl, especially branched C5-C 15 Alkyl, especially branched C 10 ~C 15 In certain embodiments, R 6 But branched C 10 Alkyl, in particular 5-ethyl-6-methylheptan-2-yl.

[0169] In an embodiment in which the surfactant is represented by formula (III), C of formula (III) n H 2n+1 The alkyl group is unsubstituted and may be linear or branched. n H 2n+1 The group is a linear alkyl. The value n may be from 12 to 100, in particular from 12 to 80, in particular from 12 to 50, in particular from 12 to 30.

[0170] How to use The present invention suppresses, inhibits or prevents aggregation of proteins in liquid formulations. Thereby, the stability of protein-containing formulations is increased and the amount of undesirable aggregates and / or degradation products is reduced or prevented. In view of this, the present invention provides in a further aspect the use of an amphiphilic surfactant as a stabilizer for protein-containing formulations. Preferably, the amphiphilic surfactant is a vitamin E analog, a sitosterol analog or a poly(alkylene glycol) alkyl ether analog.

[0171] In certain embodiments, the amphiphilic surfactant is an amphiphilic antioxidant surfactant, particularly a vitamin E analog.

[0172] In certain embodiments, the hydrophilic portion of the amphiphilic surfactant comprises a poly(alkylene glycol) moiety, particularly a poly(C1-C4 alkylene glycol) moiety, such as a poly(ethylene glycol) (PEG) moiety or a poly(propylene glycol) moiety. The poly(alkylene glycol) moiety, such as a poly(C1-C4 alkylene glycol) moiety, particularly a poly(ethylene glycol) moiety, may have an average molecular weight in the range of about 100 to about 10,000 g / mol, particularly in the range of about 300 to about 3,000 g / mol, particularly in the range of about 400 to about 2,000 g / mol.

[0173] In certain embodiments, the hydrophilic portion of the amphiphilic surfactant may comprise a moiety other than PEG, such as dimethylisosorbide oligomers, cellulose oligomers, polysarcosine oligomers, PAS oligomers ("naturally disordered biosynthetic polymers made of small L-amino acids Pro, Ala and / or Ser" see e.g. PASylation®: A versatile technology to extend drug delivery, Binder et al. Current Opinion in Colloid and Interface Science 31 (2017) pg. 10-17) or other readily biodegradable oligomers of small biorenewable materials. Thus, in formula (I)-i, (I), (II)-i, (II) and / or (III), R 1 Alternatively, it may be a dimethyl isosorbide oligomer, a cellulose oligomer, a polysarcosine oligomer or a PAS oligomer.

[0174] In certain embodiments, the amphiphilic surfactant has formula (I)-i: [ka] or Formula (II)-i: [ka] or Formula (III): [ka] (In the formula: Regarding formula (I)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 2 , R 3 , R4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (II)-i: L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; R 4 and R 5 each independently represents hydrogen and C1-C 10 alkyl; R 6 But C5~C 20 is alkyl; Regarding formula (III): L 1 is a linear or branched dicarboxylic acid linking group; R 1 is a poly(C1-C4 alkylene glycol) group, e.g., a PEG group, optionally wherein the poly(C1-C4 alkylene glycol) group is terminated with a hydroxyl or a C1-C6 alkoxyl; and n is 12 to 100, for example, 12 to 80.

[0175] In embodiments where the surfactant is a vitamin E analogue, for example represented by formula (I)-i or (I), the surfactant is in particular derived from vitamin E, in particular tocopherol, in particular α-tocopherol. The surfactant can be any amphiphilic surfactant present in an amount sufficient to improve protein stability against aggregation. In certain embodiments, the amphiphilic surfactant has a molecular weight of 10,000 g / mol or less, in particular 7,500 g / mol or less, 5,000 g / mol or less, 3,000 g / mol or less, 2,500 g / mol or less, or even 2,000 g / mol or less.

[0176] Some examples of surfactants include tocopherol polyethylene glycol succinate (TPGS), particularly DL-α-tocopherol polyethylene glycol succinate, such as TPGS-750-M, TPGS-1000, TPGS-1500, TPGS-400, TPGS-1100-M, TPGS-2000, TPGS-860-oleate, TPGS-PEG-PPG-PEG-1100 and TPGS-PPG-PEG-70-butyl, and DL-α-tocopherol polypropylene glycol succinate, such as TPPG-1000 and TPPG-1000-butyl; and polyethylene glycol α-tocopherol diester of sebacic acid (PTS), such as PTS-600. In certain embodiments, the vitamin E analog is of formula (I) as described in any one of embodiments 1-30, 52, and 53.

[0177] In embodiments where the surfactant is a sitosterol analogue, for example represented by formula (II)-i or (II), the surfactant is particularly from the plant sterol family, particularly sitosterol, especially β-sitosterol. Some examples of surfactants include β-sitosterol methoxyethylene glycol succinate (Nok), particularly Nok with mPEG, such as mPEG550. In certain embodiments, the sitosterol analogue is of formula (II) as described in any one of embodiments 1-17, 31-43, and 54-55.

[0178] In embodiments where the surfactant is a poly(alkylene glycol) alkyl ether analog, for example represented by formula (III), the surfactant is in particular derived from a poly(ethylene glycol) alkyl ether. Some examples of surfactants include analogs of Brij surfactants, in particular analogs of Brij 30, Brij 35, Brij 52, Brij 56, Brij 58, Brij 72, Brij 76, Brij 78, Brij 92, Brij 96, Brij 98 and analogs thereof. Preferably, the poly(alkylene glycol) alkyl ether analog is selected from Brij 30, Brij 35, Brij 52, Brij 56, Brij 58, Brij 72, Brij 76, Brij 78, Brij 92, Brij 96, and Brij 98. In certain embodiments, the poly(alkylene glycol) alkyl ether analog is an analog of Brij 58. In certain embodiments, the poly(alkylene glycol) alkyl ether analog is of formula (III) as described in any one of embodiments 1-17, and 44-51.

[0179] The surfactant compound represented by formula (I)-i or (II)-i or (III) is a compound represented by the residue R 1 The poly(C1-C4 alkylene glycol) group, for example, PEG, may have an average molecular weight in the range of about 100 to about 10,000 g / mol, particularly in the range of about 200 to about 5,000 g / mol, particularly in the range of about 250 to 2500 g / mol, about 400 to about 2,000 g / mol, or about 500 to 1500 g / mol, for example, about 550, about 750, or about 1000 g / mol.

[0180] residue R 1 The poly(C1-C4 alkylene glycol) group is 1 The linking group can be a linear or branched dicarboxylic acid or anhydride that forms an ester bond to the core structure and the poly(C1-C4 alkylene glycol) group. In particular, L 1The branched dicarboxylic acid linking group of formula (III) contains at least one carbon atom bonded to two C1-C4 alkyl groups, preferably two methyl substituents. Exemplary branched or linear dicarboxylic acid linking groups have 2-20 carbon atoms, such as succinic acid, sebacic acid, malonic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, 2,2-dimethylsuccinic acid, 2,2-dimethylglutaric acid, and 3,3-dimethylglutaric acid. In one embodiment, the surfactant of formula (III) is not a diester of malonic acid.

[0181] In certain embodiments, R 1 further comprises an end group bonded to the end of the poly(C1-C4 alkylene glycol) group. The end group is in particular bonded to the poly(C1-C4 alkylene glycol) group via an ether, ester or amide bond, preferably the end group is in particular bonded to the poly(C1-C4 alkylene glycol) group via an ether bond. The end group is in particular a hydroxyl or C1-C6 alkoxyl group, in particular a methoxyl (-OCH3) group.

[0182] R 2 , R 3 , R 4 and R 5 are independently hydrogen or C1-C 10 alkyl, in particular hydrogen, methyl or ethyl, in particular hydrogen or methyl. In certain embodiments, R 5 is methyl. In certain embodiments, R 4 is methyl. In certain embodiments, R 4 and R 5 is methyl. In a further embodiment, R 2 and R 3 is independently hydrogen or methyl. In certain embodiments, R 4 and R 5 is methyl and R 2 and R 3 is independently hydrogen or methyl. In certain embodiments where the surfactant has formula (I)-i, R 2 , R 3 , R4 and R 5 are all methyl. In certain embodiments where the surfactant has formula (II)-ii, R 4 and R 5 Both are methyl.

[0183] R 6 But C5~C 20 It is an alkyl group. 6 may be linear or branched and unsubstituted. In particular, R 6 is branched.

[0184] In embodiments where the surfactant has formula (I)-i, R 6 But C5~C 20 Alkyl, especially C 10 ~C 15 Alkyl, especially branched C 10 ~C 15 In certain embodiments, R 6 But branched C 15 alkyl, in particular 4,8,12-trimethyltridecyl. In certain embodiments, R 5 and R 6 The carbon atom bonded to has the R configuration.

[0185] In embodiments where the surfactant is represented by formula (II)-i, R 6 But C5~C 20 Alkyl, especially branched C5-C 15 Alkyl, especially branched C 10 ~C 15 In certain embodiments, R 6 But branched C 10 Alkyl, in particular 5-ethyl-6-methylheptan-2-yl.

[0186] In an embodiment in which the surfactant is represented by formula (III), C of formula (III) n H 2n+1 The alkyl group is unsubstituted and may be linear or branched. n H 2n+1The group is a linear alkyl. The value n may be from 12 to 100, in particular from 12 to 80, in particular from 12 to 50, in particular from 12 to 30.

[0187] In a further aspect, the present invention provides a method for improving the stability of a formulation comprising a protein, comprising incorporating an amphiphilic surfactant into a solution, wherein the amphiphilic surfactant component is present in an amount sufficient to improve the stability of the surfactant against aggregation.In certain embodiments, the method comprises contacting a protein with at least one amphiphilic surfactant to produce a liquid formulation comprising the protein and the amphiphilic surfactant, thereby stabilizing the protein.All of the above embodiments in this section relating to the use of an amphiphilic surfactant as a stabilizer for protein-containing formulations are equally applicable to the above method.

[0188] In a further aspect, the present invention provides a method for preventing or inhibiting protein aggregate formation in a protein-containing formulation, particularly by using an amphiphilic antioxidant surfactant as a stabilizer in the formulation, for example to prevent or inhibit protein aggregate formation during frozen or liquid storage of the formulation. All of the above embodiments in this section relating to the use of an amphiphilic surfactant as a stabilizer for a protein-containing formulation are equally applicable to the above method.

[0189] Furthermore, the present invention provides the use of an amphiphilic surfactant in the manufacture of a stable protein-containing formulation. All of the above embodiments in this section relating to the use of an amphiphilic surfactant as a stabilizer for a protein-containing formulation are equally applicable to the above uses.

[0190] Protein-containing formulations containing amphiphilic surfactants The present invention provides a protein-containing formulation comprising an amphiphilic surfactant, the amphiphilic surfactant being present in an amount sufficient to improve the stability of the protein against aggregation. The amphiphilic surfactant used in the protein-containing formulation of the present invention is not derived from a fatty acid building block, e.g., a polysorbate.

[0191] Thus, the present invention provides stable protein-containing formulations comprising amphiphilic surfactants.

[0192] The protein may be an antibody (e.g., ADC, mAb, Fab), an intrinsically disordered protein, or an enzyme. The protein is preferably an antibody.

[0193] The antibodies used in the present invention are not particularly limited, so long as they bind to the antigen of interest.

[0194] The immunoglobulin class of antibodies that may be used in the formulations of the present invention is not particularly limited; the class may be any class, including IgG, such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM.

[0195] Antibodies that may be used in the formulations of the present invention may also include whole antibodies, as well as antibody fragments, such as Fv, Fab, and F(ab)2, as well as minibodies (low molecular weight antibodies), such as monovalent or bivalent single chain Fvs (such as scFv, sc(Fv)2, diabodies such as scFv dimers) obtained by linking antibody variable regions via a linker, such as a peptide linker.

[0196] The above-mentioned antibodies that can be used in the present invention can be prepared by methods known to those skilled in the art.

[0197] In some embodiments, the antibody is a monoclonal antibody. The monoclonal antibody that can be used in the present invention includes not only those derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified genetically modified antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. The antibody can also include genetically modified antibodies obtained by artificially modifying antibody constant regions to change the physical properties of antibody molecules (specifically, changing isoelectric point (pI), improving affinity for Fc receptors, etc.) to improve blood persistence and in vivo pharmacokinetics.

[0198] In some embodiments, the antibody is an antigen-binding fragment. For example, the antigen-binding fragment may include one or more of a single chain variable fragment (scFv), a single domain antibody, a single chain antibody, and a heavy and / or light chain of a Fab. The antigen-binding fragment may also be a Fab, such as a Fab with a modified light chain constant region or an unmodified Fab. In some embodiments, the protein is an antigen-binding fragment. The Fab or other antigen-binding compound that may be used in the present invention may be derived from a single copy or clone, including any eukaryotic, prokaryotic, or phage clone (e.g., a monoclonal antibody (mAb)). The antibody, Fab, or other antigen-binding compound, or the nucleic acid encoding it, may be provided in an isolated form.

[0199] Monoclonal antibody-producing hybridomas can be prepared by conventional methods described below. Specifically, immunization is performed by conventional immunization methods using a desired antigen or cells expressing a desired antigen as a sensitizing antigen. The prepared immune cells are fused with known parent cells by conventional cell fusion methods. The fused cells are screened for monoclonal antibody-producing cells (hybridomas) by conventional screening methods. Hybridomas can be generated, for example, according to the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73:3-46). If the antigen has low immunogenicity, immunization can be performed with the antigen linked to an immunogenic macromolecule such as albumin.

[0200] Alternatively, it is possible to use recombinant antibodies produced using recombinant gene technology, in which antibody genes are cloned from hybridomas and inserted into a suitable vector, and the resulting vector is introduced into a host (see, for example, Carl, AK Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies, Published in the United Kingdom by Macmillan Publishers, 1990). Specifically, cDNA for an antibody variable region (V region) is synthesized from the mRNA of a hybridoma using reverse transcriptase. When a DNA encoding an antibody V region of interest is obtained, the DNA is ligated to a DNA encoding a desired antibody constant region (C region). The resulting construct is inserted into an expression vector. Alternatively, the DNA encoding the antibody V region can be inserted into an expression vector carrying the DNA of the antibody C region. The resulting construct is inserted into an expression vector so that it is expressed under the control of an expression regulatory region, e.g., an enhancer and a promoter. A host cell is then transformed with the expression vector to express the antibody.

[0201] In the present invention, artificially modified genetically engineered antibodies, such as chimeric and humanized antibodies, can be used to reduce heterologous antigenicity to humans. Such modified antibodies can be produced using known methods. A chimeric antibody is an antibody that has the heavy and light chain variable regions of an antibody from a non-human mammal, such as a mouse, and the heavy and light chain constant regions of a human antibody. A chimeric antibody can be produced by ligating the DNA encoding the variable region of a mouse antibody to the DNA encoding the constant region of a human antibody, inserting the ligate into an expression vector, and then introducing the vector into a host for expression.

[0202] Humanized antibodies, also called reshaped human antibodies, are obtained by replacing the complementarity determining regions (CDRs) of a human antibody with those of an antibody derived from a non-human mammal, e.g., a mouse. Conventional recombinant gene techniques are known. Specifically, a DNA sequence is synthesized by PCR using several oligonucleotides designed to have mouse antibody CDRs linked to human antibody framework (FR) regions and prepared to have overlapping regions at the ends. The resulting DNA is ligated to DNA encoding human antibody constant regions and then inserted into an expression vector. The expression vector is introduced into a host to produce a humanized antibody (see EP 239400 and WO 96 / 02576). The human antibody FRs linked by the CDRs are selected such that the complementarity determining regions form a preferred antigen-binding domain. Amino acids in the framework regions of the antibody variable region can be substituted as necessary so that the complementarity determining regions of the reshaped human antibody form a suitable antigen-binding domain (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0203] Methods for obtaining human antibodies are also known. For example, a desired human antibody having antigen-binding activity can be obtained by sensitizing human lymphocytes in vitro with an antigen of interest or cells expressing the antigen of interest; fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese Patent Application Publication No. (JP-B) 01-59878 (examined approved Japanese patent application published for opposition)). Alternatively, a desired human antibody can also be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with a given antigen (see WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735). Furthermore, techniques are known for obtaining human antibodies by panning using human antibody libraries. For example, the variable regions of human antibodies can be expressed as single chain antibodies (scFvs) on the surface of phages using phage display methods, and then phages that bind to antigens can be selected. The genes of the selected phages can be analyzed to determine the DNA sequences that code for the variable regions of human antibodies that bind to antigens. When the DNA sequences of scFvs that bind to antigens are identified, appropriate expression vectors carrying these sequences are constructed to obtain human antibodies. Such methods are already well known. See WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, and WO 95 / 15388. The antibodies used in the present invention also include such human antibodies.

[0204] When the antibody gene is isolated and introduced into a suitable host to produce the antibody, the host and expression vector can be used in a suitable combination. When a eukaryotic cell is used as the host, animal cells, plant cells, and fungal cells can be used. Animal cells include (1) mammalian cells, such as CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cells, such as Xenopus oocytes; and (3) insect cells, such as sf9, sf21, and Tn5. Known plant cells include cells derived from Nicotiana, such as Nicotiana tabacum, which can be cultured as callus. Known fungal cells include yeasts, such as Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi, such as Aspergillus, e.g., Aspergillus niger. When using prokaryotic cells, production systems using bacterial cells can be used. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. Antibodies can be obtained by introducing the antibody gene of interest into these cells by transformation, and then culturing the transformed cells in vitro.

[0205] There are known techniques for substituting amino acids in antibodies to improve antibody activity, physical properties, pharmacokinetics, safety, etc. Examples of such techniques are described below. The antibodies of the present invention also include those having such amino acid substitutions.

[0206] Techniques for replacing amino acids in IgG antibody variable regions have been reported, including humanization (Tsurushita N, Hinton PR, Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax., Methods. 2005 May;36(1):69-83); affinity maturation to enhance binding activity by amino acid replacement in the complementarity determining region (CDR) (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci USA. 2005 Jun 14;102(24):8466-71); and improvement of physicochemical stability by amino acid replacement in the framework (FR) (Ewert S, Honegger A, Pluckthun A., Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable There are also known techniques for enhancing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by substituting amino acids in the IgG antibody Fc domain (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review).Furthermore, in addition to techniques for enhancing effector functions, reports have been published on techniques for improving antibody half-life in blood by substituting amino acids in Fc (Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176(1): 346-56; Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15(7): 637-40). Other known techniques include an amino acid substitution technique for controlling the isoelectric point (pI) of an antibody to improve blood persistence or in vivo pharmacokinetics, specifically, a technique for modifying amino acid residues exposed on the surface of an antibody to control the pI of the antibody (WO 07 / 114319).Various techniques for substituting amino acids in the constant region to improve the physical properties of an antibody are also known (WO 09 / 41613).

[0207] In some embodiments, the protein is a disordered protein. When the disordered protein gene is isolated and introduced into a suitable host to produce the disordered protein, the host and expression vector can be used in an appropriate combination. When a eukaryotic cell is used as a host, animal cells, plant cells, and fungal cells can be used. Animal cells include (1) mammalian cells, such as CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cells, such as Xenopus oocytes; and (3) insect cells, such as sf9, sf21, and Tn5. Known plant cells include cells from Nicotiana, such as Nicotiana tabacum, that can be cultured as callus. Known fungal cells include yeasts, such as Saccharomyces, for example Saccharomyces cerevisiae, and filamentous fungi, such as Aspergillus, for example Aspergillus niger.

[0208] The concentration of a protein, such as an antibody (e.g., ADC, mAb, Fab), an intrinsically denatured protein, or an enzyme, in the protein-containing formulation of the present invention is not particularly limited. The protein concentration is preferably 5 mg / ml or more, 10 mg / ml or more, 25 mg / ml or more, 50 mg / ml or more, more preferably 100 mg / ml or more, even more preferably 120 mg / ml or more, even more preferably 150 mg / ml or more, and even more preferably 180 mg / ml or more. The upper limit of the antibody concentration in the formulation of the present invention is not particularly limited; the upper limit is generally 300 mg / ml. The protein concentration can be 1 to 300 mg / ml, for example, 1 to 250 mg / ml.

[0209] The stability of the protein formulation of the present invention may include evaluating the chemical stability, physical stability or functional stability of the protein and / or surfactant. This may be measured, for example, by using forced degradation assays. The formulation of the present invention typically exhibits a high level of protein stability. Thus, the protein in the formulation may retain an acceptable degree of chemical structure or biological function after storage under the exemplary conditions defined herein. A formulation may be stable even if the protein contained therein does not maintain 100% of its chemical structure or biological function after storage for a given time. Under certain circumstances, the maintenance of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the protein structure or function after storage for a given time may be considered to be "stable".

[0210] Stability can be measured, inter alia, by determining the percentage of native protein remaining in the formulation after storage at a given temperature for a given time. The percentage of native protein can be determined, inter alia, by size-exclusion chromatography, (e.g., size-exclusion high performance liquid chromatography (SE-HPLC)), where native means non-aggregated and non-degraded. "Acceptable stability" as used herein means that at least 90% of the native form of the protein can be detected in the formulation after storage at a given temperature for a given time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the protein can be detected in the formulation after storage at a given temperature for a given time. The predetermined time period after which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more.

[0211] In assessing stability, the temperature at which the pharmaceutical formulation may be stored may be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, a pharmaceutical formulation may be considered stable if greater than about 95%, 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 6 months of storage at room temperature. A pharmaceutical formulation may also be considered stable if greater than about 94%, 95%, 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 6 months of storage at room temperature. A pharmaceutical formulation may also be considered stable if greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 28 days of storage at 45° C. A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 3 months of storage at −20° C. A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 3 months of storage at −30° C. A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native protein is detected by SE-HPLC after 3 months of storage at −80° C.

[0212] Stability may also be measured, inter alia, by determining the concentration of particles present in the protein-containing formulation. The concentration of particles may be determined, inter alia, by microflow imaging (MFI) or light obscuration (LO). There are many proteinaceous and non-proteinaceous particle classifications, depending on criteria such as size, source, or composition. From an analytical point of view, classification by size is the main attribute that characterizes particles. Particles are defined as materials having a size greater than 0.1 μm, and are further classified into non-visible (0.1-100 μm) and visible particles (greater than 100 μm). Submicron particles (0.1-1 μm) are a subgroup of non-visible. Protein particles are a subset of protein aggregates. In certain embodiments, less than about 6000 particles ≧10 μm and / or less than 600 particles ≧25 μm per 100 mL or less total volume (small volume parenteral formulation (SVP)) as measured by light obscuration methods are in accordance with the USP <787> and USP <788> can be detected in the formulation after storage at a given temperature for a given time, as defined in

[0213] In certain embodiments, less than about 25 particles per mL > 10 μm and / or less than 3 particles per mL > 25 μm for a total volume of 100 mL or more (large volume parenteral (LVP)) as measured by light obscuration methods, as defined by the USP <787> and USP <788> can be detected in the formulation after storage at a given temperature for a given time, as defined in

[0214] The predetermined time period after which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more.

[0215] In assessing stability, the temperature at which the pharmaceutical formulation may be stored may be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, the pharmaceutical formulation may be considered stable if less than about 6000, 5000, 4000, 3000, 2000, 1000 particles > 10 μm and / or less than about 600, 500, 400, 300, 200, 100 particles > 25 μm in a volume of 100 mL are detected by light obscuration methods after 6 months of storage at 5°C. A pharmaceutical formulation may also be considered stable if, after 6 months of storage at room temperature, less than about 6000, 5000, 4000, 3000 particles ≧10 μm and / or less than about 600, 500, 400, 300 particles ≧25 μm in a volume of 100 mL are detected by light obscuration methods. A pharmaceutical formulation may also be considered stable if, after 28 days of storage at 45° C., less than about 6000, 5000, 4000 particles ≧10 μm and / or less than about 600, 500, 400 particles ≧25 μm in a volume of 100 mL are detected by light obscuration methods. A pharmaceutical formulation may also be considered stable if less than about 6000, 5000, 4000, 3000, 2000, 1000 particles ≧10 μm and / or less than about 600, 500, 400, 300, 200, 100 particles ≧25 μm in a volume of 100 mL are detected by light obscuration methods after 3 months of storage at −20° C. A pharmaceutical formulation may also be considered stable if less than about 6000, 5000, 4000, 3000 particles ≧10 μm and / or less than about 600, 500, 400, 300 particles ≧25 μm in a volume of 100 mL are detected by light obscuration methods after 3 months of storage at −30° C. A pharmaceutical formulation may also be considered stable if less than about 6000, 5000, 4000, 3000, 2000, 1000 particles ≧10 μm and / or less than about 600, 500, 400, 300, 200, 100 particles ≧25 μm in a 100 mL volume are detected by light obscuration methods after 3 months of storage at −80° C.

[0216] Other methods, such as, for example, differential scanning calorimetry (DSC) to determine thermal stability, controlled stirring to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidity, can be used to assess the stability of the formulations of the invention. For example, a formulation of the invention can be considered stable if, after storage at about 5° C. to about 25° C. for 6 months or more, the change in optical density at 405 nm (OD405) of the formulation is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less) from the OD405 of the formulation at time zero. Measurement of the biological activity of the protein or binding affinity to its target can also be used to assess stability. For example, a formulation of the invention can be considered stable if, after storage at, for example, 5° C., 25° C., 45° C., etc. for a given period of time (e.g., 1 to 12 months), the protein contained in the formulation binds to its target with an affinity that is at least 90%, 95%, or greater than the binding affinity of the protein prior to said storage. Binding affinity can be determined, for example, by ELISA or surface plasmon resonance. Biological activity can be determined, for example, by protein activity assays, such as contacting cells expressing the protein with a formulation containing the protein. The binding of the protein to such cells can be measured directly, for example, by FACS analysis. Alternatively, the downstream activity of the protein system can be measured in the presence of the protein and compared to the activity of the protein system in the absence of the protein. Further methods for evaluating the stability of a protein in a formulation are demonstrated in the examples presented herein.

[0217] The surfactant in the formulation of the present invention can be any surfactant present in an amount sufficient to improve the stability of the protein against aggregation. In certain embodiments, the amphiphilic surfactant has a molecular weight of 10,000 g / mol or less, in particular 7,500 g / mol or less, 5,000 g / mol or less, 3,000 g / mol or less, 2,500 g / mol or less, or even 2,000 g / mol or less.

[0218] In certain embodiments, the hydrophilic portion of the amphiphilic surfactant present in the formulation comprises a polyalkylene glycol portion, in particular a polyethylene glycol portion or a polypropylene glycol portion. The polyalkylene portion, in particular the polyethylene glycol portion, may have an average molecular weight in the range of about 100 to about 10,000 g / mol, in particular in the range of about 300 to about 3,000 g / mol, in particular in the range of about 400 to about 2,000 g / mol. Some examples of surfactants that include a polyalkylene glycol moiety include tocopherol polyethylene glycol succinates (TPGS), in particular DL-α-tocopherol polyethylene glycol succinates, such as TPGS-750-M, TPGS-1000, TPGS-1500, TPGS-400, TPGS-1100-M, TPGS-2000, TPGS-860-oleate, TPGS-PEG-PPG-PEG-1100 and TPGS-PPG-PEG-70-butyl, and DL-α-tocopherol polypropylene glycol succinates, such as TPPG-1000 and TPPG-1000-butyl and derivatives thereof; and polyethylene glycol α-tocopherol diesters of sebacic acid (PTS), such as PTS-600; polyethylene glycol alkyl ether surfactants, such as Brij surfactants, in particular Brij 30, Brij 35, Brij 52, Brij 56, Brij 58, Brij 72, Brij 76, Brij 78, Brij 92, Brij 96, Brij 98; cholesteryl PEG succinate, e.g., cholesteryl PEG 1000 succinate; β-sitosterol methoxyethylene glycol succinate (Nok), particularly Nok containing mPEG, such as mPEG 550. All of the above embodiments in the section relating to the use of amphiphilic surfactants as stabilizers for protein-containing formulations are equally applicable to protein-containing formulations containing amphiphilic surfactants.

[0219] The concentration of the surfactant in the formulation is in particular at least 0.001% (w / v), in particular at least 0.01% (w / v), in particular at least 0.05% (w / v) or at least 0.1% (w / v). In a particular embodiment, the concentration of the surfactant in the formulation is in the range of 0.001-1.0% (w / v), in particular in the range of 0.02-0.8% (w / v), 0.05-0.5% (w / v), 0.1-0.5% (w / v), for example about 0.01% (w / v), about 0.02% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.08% (w / v), about 0.1% (w / v), about 0.2% (w / v), about 0.3% (w / v) or about 0.5% (w / v). In certain embodiments, the concentration of the surfactant in the formulation is about 4-20 times, preferably 4-10 times, preferably 4, 5, 6, 10 or 20 times higher than its critical micelle concentration. In certain embodiments, the concentration of the surfactant in the formulation is at least 5, 10 or 20 times higher than its critical micelle concentration.

[0220] In some embodiments, the protein-containing formulation of the present invention may further comprise one or more excipients. Exemplary excipients include, but are not limited to, sugars, (e.g., trehalose, sucrose, maltose, mannitol, sorbitol) buffers, salts, antioxidants, preservatives, solubilizers, diluents, excipients, coloring agents, and / or flavoring agents. Such sugars include those known in the art and conventionally used in pharmaceutical formulations. Examples of such buffers, salts, antioxidants, solubilizers, preservatives, diluents, excipients, coloring agents, and flavoring agents are well known in the art.

[0221] In certain embodiments, the protein-containing formulation further comprises a buffer. The buffer should in particular be suitable for maintaining the pH of the formulation at or about neutral pH. Such pH buffers include many buffers known in the art and conventionally used in pharmaceutical formulations. In particular, the buffer is selected from the group consisting of TRIS, phosphate, citrate, acetate and ammonia. When Brij58 or the compound represented by formula (III) is used as a surfactant, succinate and histidine buffers are not preferred. The protein-containing formulation preferably has a pH at which the protein is active and stable. In certain embodiments, the pH value is within the range of 4.0 to 10.0, in particular 6.0 to 8.0, in particular 6.5 to 7.5, for example about 7.0.

[0222] In certain embodiments, the formulation further comprises an isotonicity agent, such as sodium chloride, potassium chloride or calcium chloride, preferably in an amount greater than 0.1M.

[0223] The viscosity of the liquid formulation of the present invention at room temperature (25° C.) may be 30 mPa·s or less, 20 mPa·s or less, or 15 mPa·s or less.

[0224] The formulation of the present invention may further comprise an amino acid. Such amino acids include those known in the art and conventionally used in pharmaceutical formulations, for example, those described in Remington The Science and Practice of Pharmacy, 22 nd See Ed. Pharmaceutical Press, 2013, pp. 1049-1070.

[0225] The formulations of the present invention can be administered parenterally, for example, by injection. Injection includes systemic and local administration, for example, by subcutaneous injection, intravenous injection, intramuscular injection, etc. Thus, the formulations of the present invention are particularly suitable for subcutaneous and intravenous administration.

[0226] The formulations of the present invention may also be stable aqueous solutions, in particular ready-to-use stable aqueous solutions.

[0227] A further aspect of the invention relates to a kit for use in the method of the invention. The kit may comprise an amphiphilic surfactant, e.g., an amphiphilic antioxidant surfactant, in a form suitable for stabilizing a protein. In certain embodiments, the amphiphilic surfactant is a surfactant according to the invention, represented by formula (I), (II) or (III). The kit may further comprise other components, such as a therapeutic agent, a carrier, a buffer, a container, a device for administration / contact, a composition for transformation, etc. The kit may be designed for therapeutic, diagnostic and / or research use, and the further components may be suitable for the intended use. The kit may further comprise a label and / or instructions, e.g., for stabilizing a protein and / or for patient administration. Such a label and / or instructions may include, for example, information regarding the amount, frequency and method of administration of the formulation comprising the surfactant.

[0228] Methods for Protein Stabilization Further provided is a method for stabilizing a protein. In certain embodiments, a method for stabilizing a protein is provided, comprising contacting a protein of interest with an amphiphilic surfactant to produce a liquid formulation comprising the protein and the amphiphilic surfactant, thereby stabilizing the protein. In some embodiments, the method further comprises at least partially drying the liquid composition comprising the protein and the amphiphilic surfactant. Drying of the liquid formulation can be started at any time after contacting the protein with the amphiphilic surfactant. Any method for drying a liquid formulation can be used, including but not limited to freeze drying, air drying, spray drying, spray freeze drying, vacuum drying, and / or foam drying.

[0229] All of the embodiments described above in the section describing the use of amphiphilic surfactants as stabilizers for protein-containing formulations relating to proteins and amphiphilic surfactants are equally applicable to the above methods of protein stabilization.

[0230] Method of preparation The surfactants of the present disclosure can be prepared by several methods well known to those skilled in the art of organic synthesis. By way of example, the surfactants of the present disclosure can be synthesized using the methods described below, together with methods known in the art of synthetic organic chemistry, or variations thereof as would be understood by those skilled in the art.

[0231] Generally, for example, Vitamin E surfactants of formula (I) can be prepared according to the scheme shown below.

[0232] General scheme 1 [ka] The steps depicted in general Scheme 1 involve standard esterification chemistry, the conditions of which are known to those skilled in the art.

[0233] Generally, for example, sitosterol surfactants of formula (II) can be prepared according to the scheme shown below.

[0234] General scheme 2 [ka] The steps depicted in general Scheme 2 involve standard esterification chemistry, the conditions of which are known to those skilled in the art.

[0235] General scheme 3 Generally, for example, poly(alkylene glycol) alkyl ether surfactants of formula (III) can be prepared according to the scheme shown below. [ka]

[0236] The steps depicted in general Scheme 3 involve standard esterification chemistry, the conditions of which are known to those skilled in the art.

[0237] The formulations of the invention can be prepared by mixing the protein with an amphiphilic surfactant, such as an amphiphilic antioxidant surfactant, including the step of contacting the protein with the amphiphilic surfactant to produce a formulation comprising the protein and the amphiphilic surfactant. The formulation produced is in a liquid state.

[0238] The formulations of the present invention can be prepared by mixing the protein with an amphipathic surfactant, for example a 5% (w / v) surfactant stock solution, which is prepared and sterile filtered using a 0.22 μm filter unit. The stock solution required to reach the final surfactant concentration is added to the respective protein drug substance.

[0239] Therapeutic Uses of Protein-Containing Preparations The stable protein-containing formulation of the present invention can be useful for treatment.Accordingly, the present invention can be used in a pharmaceutical formulation suitable for patient administration.In view of this, in a further aspect, the present invention provides a protein-containing formulation for use in treatment, the formulation comprises an amphiphilic surfactant, and the amphiphilic surfactant is present in an amount sufficient to improve the stability of protein against aggregation.Accordingly, the present invention provides a stable protein-containing formulation for use in treatment, the protein-containing formulation comprises an amphiphilic surfactant.

[0240] The invention further provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein-containing formulation, wherein the protein-containing formulation comprises an amphipathic antioxidant surfactant, and the amphipathic antioxidant surfactant is present in an amount sufficient to improve the stability of the protein against aggregation.

[0241] Thus, the present invention provides a method for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a stable protein-containing formulation comprising an amphiphilic surfactant.

[0242] In a further aspect, the present invention provides the use of a protein-containing formulation in therapy, wherein the formulation comprises an amphiphilic surfactant, and wherein the amphiphilic surfactant is present in an amount sufficient to improve the stability of the protein against aggregation.Thus, the present invention provides the use of a stable protein-containing formulation in therapy, wherein the protein-containing formulation comprises an amphiphilic surfactant.

[0243] In a further aspect, the present invention provides the use of a protein-containing formulation in the manufacture of a medicament for treating a disease or disorder, wherein the formulation comprises an amphiphilic surfactant, and the amphiphilic surfactant is present in an amount sufficient to improve the stability of the protein against aggregation. Thus, the present invention provides the use of a stable protein-containing formulation in the manufacture of a medicament for treating a disease or disorder, wherein the protein-containing formulation comprises an amphiphilic surfactant.

[0244] All of the embodiments described above in the section relating to protein-containing formulations comprising amphiphilic surfactants are equally applicable to the above formulations for use and methods of treatment.

[0245] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments which are given by way of illustration of the present invention and are not intended to be limiting of the present invention. EXAMPLES

[0246] Synthesis of surfactants containing the formula (I), (II) or (III) Prototypical steps: To a 250 mL round bottom flask equipped with a stir bar was added the lipophilic alcohol (1 eq) and anhydride (1 eq). Then DCM (100 mL) was added and the flask was stirred briefly. Then Et3N (3 eq) and finally DMAP (0.15 eq) were added. Upon completion, the reaction mixture was transferred to a 500 mL separatory funnel and the flask was rinsed with 50 mL of DCM. The organic layer was washed with 3×100 1M HCl solution and then with 50 mL of 10% brine. The organic phase was dried over anhydrous MgSO4, filtered and the organic solvent was removed by rotary evaporation. The product was recrystallized from about 200 mL of hot heptane, cooled to room temperature and filtered. The filter cake was washed with heptane and the product was dried in a vacuum oven.

[0247] To a 500 mL round bottom flask equipped with a stir bar was added the carboxylic acid generated above (1 equivalent), then toluene (200 mL), then PEG1000 (3 equivalents for PEG, 1 equivalent for PEG-OMe). The flask was briefly swirled to mix, then p-TSA was added. * HO (0.15 equiv.) was added. The flask was placed in an oil bath heated to 140 °C and the flask was fitted with a Dean-Stark trap and condenser and brought to reflux. Upon completion, the reaction was removed from the heat and the solvent was removed by rotary evaporation. The crude product was taken up in deionized HO (350 mL) and the volatile organic solvents were removed by distillation. The aqueous phase was then extracted with DCM (300 mL) (with gentle stirring to prevent emulsions) to give the crude product. The crude product was purified by column chromatography on silica and the liquid packing technique (the crude product was taken up in EtOAc for liquid packing). The column was run first with a 50:50 (EtOAc / Hexanes) mixture, then DCM, then a gradient of 0-4% MeOH / DCM.

[0248] Poly(alkylene glycol) alkyl ether analogues Example 1: [ka] 1H NMR (400MHz, クロロホルム-d)δ 4.27-4.20(m,2H), 4.07(t,J=6.8Hz,2H), 3.85-3.44(m,87H), 2.76(t,J=6.0H z, 1H), 2.68-2.58(m,4H), 1.67-1.56(m,2H), 1.26(s,27H), 0.92-0.83(m,3H). 13C NMR(101MHz, CDCl3)δ 172.14, 172.12, 72.46, 70.54, 70.49, 70.29, 68.96, 64.76, 63.71, 61.59, 31.80, 29.56, 29 .54, 29.52, 29.51, 29.45, 29.39, 29.22, 29.13, 29.02, 28.99, 28.49, 25.77, 22.56, 14.01.

[0249] Example 2:

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[0250] Example 3:

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[0251] Example 4:

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[0252] Example 5:

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[0253] Example 6:

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[0254] Example 7:

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[0255] Example 8:

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[0256] ビタミンE analog Example 9:

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[0257] Example 10:

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[0258] Example 11:

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[0259] Example 12:

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[0260] Example 13:

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[0261] Example 14:

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[0262] Example 15: [ka] 1H-NMR (DMSO-d6, 400MHz):δ 4.16-4.13(m,2H), 3.58-3.50(m,2H), 3.49-3.43(m,53H), 3.42-3.41(111, 2H), 3.24(s,3H), 2.75(s,2H), 2.54-2.51(m,2H), 2.50 -2.49(m,2H), 2.00(s,3H), 1.90(d,J=9.2Hz, 6H), 1.79-1.65(m,2H), 1.50-1.47(m,3H), 1.36-1.01(m,28H), 0.88-0.79(m,12H)ppm

[0263] Example 16: [ka]

[0264] Surfactant formulation data Example 18: Preparation of formulations according to the present invention All surfactants were solubilized in water for injection (WFI) to produce a 5% (w / v) stock solution. The solution was sterile filtered through a 0.22 μm filter unit. The stock solution was stored at −20° C. until use. In this section, the following proteins were used to demonstrate the broad applicability of the present invention across several protein classes: Protein 1 is a disordered protein; Protein 2 is a fab and Protein 3 is a monoclonal antibody.

[0265] 18.1: Formulations containing disordered proteins: Preparation of protein 1 Buffer solutions corresponding to the APIs were prepared using 20 mM histidine, 220 mM sucrose pH 5.8 in water. The respective detergent stock solutions were added appropriately to the buffer to reach the final detergent concentrations (Brij58 succinate 0.01% (w / v); Vit E variant 0.04% (w / v); SPGS-550-M 0.01% (w / v)).

[0266] 18.2: Preparation of fab-containing formulation: protein 2 Buffer solutions corresponding to the API were prepared with 10 mM histidine in water at pH 5. The respective detergent stock solutions were added appropriately to the buffer to reach the final detergent concentrations (Brij58 succinate 0.01% (w / v); VitE variants 0.04% (w / v); SPGS-550-M 0.01% (w / v)).

[0267] 18.3: Monoclonal antibody preparations: Preparation of protein 3 Buffer solutions corresponding to the API were prepared with 3.05 mg / mL adipic acid in water at pH 7. The respective surfactant stock solutions were added appropriately to the buffer to reach the final surfactant concentrations (Brij58 succinate 0.01% (w / v); VitE variants 0.04% (w / v); SPGS-550-M 0.01% (w / v)).

[0268] Example 19: Mechanism of action of amphiphilic surfactants in protein-containing liquid formulations 19.1 Purpose: The objective of this experiment was to investigate the type of stabilization mechanism used by surfactants in protein-containing formulations. A Pendant-Drop Tensiometer was used to determine protein (API) displacement by surfactants at the air / water interface. Surfactant interfacial saturation reduces the risk of interfacial stress on the protein and the associated adverse effects potentially affecting protein quality attributes.

[0269] 19.2 Sample preparation: The corresponding buffer solutions for the proteins were prepared and sterile filtered using a 0.22 μm filter unit. The appropriate detergent was added to the buffer solution using a 5% stock solution as described above. The buffer detergent mixture was divided equally into two Nalgene bottles. To one of the two bottles, the respective API was added to reach a final concentration of 1 mg / mL for protein 1 and 10 mg / mL for proteins 2 and 3.

[0270] 19.3 Method: Equilibrium surface tension was measured using a PAT-1M tensiometer (S-interface). The system was rinsed 12 times with the sample to be measured. Hanging drops of constant volume (20 μL) were measured every second for 60 minutes. Drug substance samples with and without surfactant were measured on the same day to avoid operator, sample and day-to-day variability.

[0271] 19.4 Results: 19.4.1 Vitamin E derivatives VEDS = Vitamin E Dimethyl Succinate (Example 10); VEDG2.2 = Vitamin E Dimethyl Glutarate (Example 12)

[0272] Line (a) in Figures 1A-1C shows that protein 1 without surfactant demonstrates surface activity as the surface tension decreases over time. Formulations with surfactant TPGS1000 in Figure 1A and VEDS and VEDG2.2 in Figures 1B and 1C show even higher surface activity, resulting in a significant reduction in surface tension. Thus, the surfactant displaces the protein from the interface, relieving the interfacial stress.

[0273] 1A-1C show the displacement of protein 1. The same mechanism holds for protein 2 (Fab) and protein 3 (mAb). The data suggest that amphiphilic surfactants, such as vitamin E surfactants, act primarily by a displacement mechanism, as the surfactant displaces the protein from the interface due to its higher surface activity resulting in lower surface tension (line (c)).

[0274] 19.4.2 Sitosterol derivatives As shown by FIG. 1D, line (a), protein 1 without surfactant is shown to be surface active as the surface tension decreases over time. Formulations containing SPGS-550-M (NOK) surfactant in the absence or presence of protein 1 (lines (b) and (c) respectively) show even higher surface activity, resulting in a significant reduction in surface tension. Thus, amphiphilic surfactants, such as NOK, displace protein from the interface reducing interfacial stress.

[0275] FIG. 1D shows the displacement of a minimal model protein 1. The same mechanism is true for protein 2 (Fab) and protein 3 (mAb). The data suggest that amphiphilic surfactants, such as sitosterol derivatives such as SPGS-550-M, act by a displacement mechanism, since the surfactant displaces the protein from the interface due to its higher surface activity resulting in a lower surface tension (line (c)). As the surface tension does not decrease to a surface tension level of about 40 mN / m, it cannot be excluded that this surfactant also employs a selective binding mechanism, since it has a lower surface activity compared to polysorbate.

[0276] 19.4.3 Poly(alkylene glycol) alkyl ether derivatives Line (a) of Figures 1E and 1F shows that protein 1 without surfactant shows to be surface active as the surface tension decreases over time. Formulations with the surfactants Brij 58 and Brij 58 succinate in the absence or presence of protein 1 (lines (b) and (c) respectively) show even higher surface activity, resulting in a significant reduction in surface tension. Thus, the surfactant displaces the protein from the interface relieving interfacial stress.

[0277] 1E and 1F show the displacement of a minimal model protein 1. The same mechanism holds for protein 2 (Fab) and protein 3 (mAb). The data suggest that amphipathic surfactants, such as Brij 58 and Brij 58 succinate, act primarily by a displacement mechanism, as the surfactant displaces the protein from the interface due to its higher surface activity resulting in lower surface tension (line (c)).

[0278] Example 20: Evaluation of the stabilizing effect of amphiphilic surfactants as protein (API) stabilizers in liquid formulations 20.1 Purpose: The objective of this experiment was to investigate the effectiveness of amphiphilic surfactants to protect proteins from interfacial stresses, such as those present during production, shipping and application. As a surrogate for this stress, a forced disintegration assay (orbital shaking) applying mechanical stress was used to determine whether selected surfactants stabilize (reduce particle formation (protein aggregates)) three different protein APIs (disordered protein, Fab, mAb) against mechanical stress.

[0279] 20.2 Sample preparation: To each protein API was added the respective amount of detergent (5% stock solution) required to reach the final detergent concentration (Brij58 0.01%; VitE variants 0.04%; SPGS-550-M 0.01%) as previously described. 1.2 mL of sample was aliquoted into each 6R vial (2x). Vials of the same sample were pooled after testing to generate sufficient volume for all analytical tests.

[0280] 20.3 Method: Mechanical stress was generated by orbital shaking at 250 rpm using an orbital shaker at ambient temperature for 0, 7, 24 and 48 hours. Samples were subsequently analyzed by microflow imaging (MFI). Particle counts > 2 μm / mL were visualized by MFI.

[0281] 20.4 Results: 20.4.1 Prescreening of Commercial Surfactants in Comparison with Polysorbates The commercial surfactants Brij58, SPGS-550-M and TPGS-1000 were tested against the current representative standard PS20 / 80 (polysorbate 20 and polysorbate 80) as shown in Figures 2A-2C. The data shown in Figures 2A-2C show that all amphiphilic surfactants, regardless of protein class, resulted in fewer particles than the current standard (PS20 / 80). Thus, amphiphilic surfactants not derived from fatty acid building blocks, such as vitamin E, sitosterol and poly(alkylene glycol) alkyl ether derivatives, have improved stabilizing effects in protein-containing formulations than polysorbates.

[0282] 20.4.2 Vitamin E derivatives VEDS = Vitamin E Dimethyl Succinate (Example 10); VEDG3.3 = Vitamin E Dimethyl Glutarate (Example 11); VEDG2.2 = Vitamin E Dimethyl Glutarate (Example 12)

[0283] Figures 2D-2F show that vitamin E derivatives prevent particle formation. For protein 1, VEDS and VEDG 2.2 perform better than TPGS-1000. Protein 2, as a representative of Fab, and protein 3, as a representative of mAb, show significantly reduced particle counts when amphiphilic surfactants are present. This demonstrates the ability of amphiphilic surfactants to stabilize APIs under mechanical stress.

[0284] 20.4.3 Sitosterol derivatives Figures 2G-2I show that sitosterol analogs such as SPGS-550-M(NOK) prevent particle formation for the protein classes tested. Protein 2, as a representative of a Fab, and protein 3, as a representative of a mAb, show a significantly reduced number of particles when surfactants are present. This demonstrates the ability of amphiphilic surfactants to stabilize APIs under mechanical stress.

[0285] 20.4.4 Poly(alkylene glycol) alkyl ether derivatives Figures 2J-2L show that poly(alkylene glycol) alkyl ether derivatives such as Brij58 and Brij58 succinate prevent particle formation. For protein 1, Brij58 succinate works better than Brij58. Protein 2, as a representative of a Fab, and protein 3, as a representative of a mAb, show a significantly reduced number of particles when amphiphilic surfactants are present. This demonstrates the ability of amphiphilic surfactants to stabilize APIs under mechanical stress.

[0286] Example 21: Evaluation of the effect of amphiphilic surfactants on stability profiles upon chemical degradation (e.g., oxidation, hydrolysis) 21.1 Purpose: Polysorbate 20 / 80, as previously described, is subject to chemical and enzymatic degradation mechanisms. To test the amphiphilic surfactants of the present disclosure for stability against oxidative and hydrolytic stress, the following forced degradation assays were performed using hydrogen peroxide and various buffers to mimic those stress conditions. The purpose was to mimic the chemical degradation pathways that the surfactants may be exposed to during shelf life.

[0287] 21.2 Method: H2O2 (0.25M and 2.5M) was added to the formulations containing surfactants and then incubated at 40°C for 6 days to mimic oxidative stress. Additionally, surfactants were added to different buffer types (e.g., histidine, citrate, phosphate and succinate buffers) and exposed to accelerated degradation stresses such as temperature stress (50°C for 1 month) at different pHs (5.5 and 6.5) to mimic chemical degradation pathways (e.g., oxidation, hydrolysis). Control samples were frozen to avoid degradation (FZ). Using chromatographic methods, the surfactant content of the formulations after accelerated conditions was determined by comparing it to the amount of surfactant added in the buffer at the beginning of the experiment.

[0288] 21.3 Results: 21.3.1 Polysorbate 80 FIG. 3A shows that PS80 is stable upon H2O2 treatment at 40° C. for 6 days under 0.25 M and 2.5 M H2O2 stress.

[0289] The results from Figure 3B show that PS80 is stable in citrate buffer regardless of pH or high temperature (e.g., 50°C for 1 month). PS80 shows reduced content for histidine, phosphate and succinate buffers at accelerated conditions, thus decomposing into free fatty acids and peroxides under those conditions.

[0290] 21.3.2 Vitamin E derivatives As shown in FIG. 3C, TPGS1000 is stable upon H2O2 treatment with 0.25M and 2.5M H2O2.

[0291] As shown in FIG. 3D, TPGS1000 is stable in all selected buffer conditions despite pH changes (pH 5.5 and pH 6.5) and high temperatures (e.g., 50° C. for 1 month).

[0292] 21.3.3 Poly(alkylene glycol) alkyl ether derivatives As shown in Figure 3E, Brij58 is stable upon H2O2 treatment. A slight decrease in Brij58 occurred in the 2.5 M H2O2 condition.

[0293] Figure 3F shows that Brij58 is stable in citrate and phosphate buffers regardless of pH or elevated temperature (e.g., 50°C for 1 month). Brij58 shows reduced content for histidine and succinate buffers at accelerated conditions.

[0294] 21.4 Summary The amphiphilic surfactants shown are believed to be stable in the presence of hydrolytic, oxidative and temperature stress in selected buffer conditions (histidine, phosphate, citrate and succinate buffers).

[0295] Example 22: Evaluation of the safety profile of amphiphilic surfactants in primary human tissues (in vitro) 22.1 Purpose: The amphiphilic nature of surfactants carries the risk of surfactant interaction and / or integration into the lipid bilayer of mammalian cells. This is a safety concern for patients, for example, when administered to humans. To demonstrate that selected surfactants are safe with respect to hemolysis, dendritic cell activation and basophil cell activation, the following assays were performed (see Methods section).

[0296] 22.2 Method: Hemolysis assay: Human whole blood was incubated with detergent for 1 hour at 37°C. Cells were spun down at 2000g for 10 minutes. The COBAS6000 biochemistry analyzer took an aliquot of the patient sample, diluted it in saline solution (0.9% sodium chloride) and measured the absorbance for hemolysis at 570nm (primary wavelength) and 600nm (secondary wavelength). From these absorbance values, the instrument calculates serum index values.

[0297] Basophil activation assay: Human whole blood was used to purify B cells using an in vitro B cell activation kit (Cellonic, Basotest, Cat#10-0500). When the correct dilutions were previously prepared, basophil cells were incubated in 100 μL of 1× washing buffer containing the final detergent concentration. Cell labeling, lysis and fixation were performed according to the manufacturer's instructions and then analyzed using a BD FORTESSA flow cytometer. Leukocytes were gated by forward and side scatter. Basophil subpopulations were gated on CD123 and IgE staining. Finally, activated basophil cells were identified by CD203c and CD63 gating. All fluorescently labeled antibodies were provided in the above kit.

[0298] Dendritic cell activation: Human whole blood was used to extract monocytes using MACS (magnetic activated cell sorting) according to the manufacturer's instructions (Miltenyi Biotec Order no. 130-114-980). Cells were incubated for 6 days in the respective differentiation medium (Miltenyi DC differentiation medium order no. 130-094-812) to yield dendritic cells. Cells were incubated for 48 hours in the corresponding detergent-containing medium and then stained for cell activation using fluorescently labeled antibodies (mouse anti-human CD14 PerCP-Cy5.5 order no. 562692 from BD to test for monocyte negativity; mouse anti-human CD11c BV605 from BD to test for dendritic cell differentiation and mouse anti-human CD80 FITC order no. 557226 from BD to test for cell activation).

[0299] 22.3 Results: 22.3.1 Vitamin E derivatives The hemolysis data shown in FIG. 4A does not demonstrate increased hemolysis potential for vitamin E derived surfactants compared to the current polysorbate standard. The B cell activation assay data shown by FIG. 4B did not show increased B cell activation of the surfactants tested compared to the positive control (activated B cells). Furthermore, VEDS, VEDG 2.2 and VEDG 3.3 perform better than TPGS1000. The data from the dendritic cell (DC) activation assay described in FIG. 4C did not show increased DC levels upon surfactant treatment. Furthermore, VEDS, VEDG 2.2 and VEDG 3.3 perform better than TPGS1000.

[0300] Therefore, it is believed that the amphiphilic surfactant represented by formula (I) does not have adverse safety effects in terms of hemolytic ability or immune system activation.It is believed that the surfactant represented by formula (I) may exhibit increased safety and / or a lower toxicological safety profile compared to commercially available vitamin E analogues.

[0301] 22.3.2 Sitosterol derivatives The hemolysis data shown in FIG. 4D does not demonstrate increased hemolytic potential for the sitosterol surfactant compared to the current polysorbate standard.

[0302] The B cell activation assay shown by FIG. 4E showed a slightly increased level of B cell activation, which is still considered safe.

[0303] Data from the dendritic cell activation assay described in FIG. 4F did not demonstrate increased DC levels upon detergent treatment.

[0304] Therefore, the amphiphilic surfactant represented by formula (II) is considered to have no adverse effects on safety in terms of hemolytic activity or immune system activation and to exhibit a safety profile equivalent to that of polysorbates.

[0305] 22.3.3 Poly(alkylene glycol) alkyl ether derivatives The hemolysis data shown in FIG. 4G demonstrates the increased hemolytic potential for Brij58 at a concentration of 0.05% (w / v). Brij58 does not contain a linker between its hydrophobic and hydrophilic moieties. By introducing a succinate linker into Brij58, its hemolytic potential was reduced to a level comparable to that of polysorbate 20. Thus, the detergent safety for Brij58 is increased by introducing a linker derived from a dicarboxylic acid, such as a succinate linker.

[0306] The B cell activation assay shown by Figure 4H did not show increased B cell activation of the tested detergents compared to the positive control (activated B cells). In accordance with the results above, basophil activation for Brij58 is reduced by introducing a linker derived from a dicarboxylic acid, such as a succinate linker.

[0307] Data from the dendritic cell activation assay described in FIG. 4I did not demonstrate increased DC levels upon detergent treatment.

[0308] Thus, it is believed that the surfactants of formula (III) do not have adverse effects on hemolysis or immune system activation. It is believed that the surfactants of formula (III) may in particular exhibit increased safety and / or a lower toxicological safety profile than other amphiphilic surfactants.

[0309] Example 23: Evaluation of the propensity of amphiphilic surfactants to inhibit platelet aggregation 23.1 Purpose: Surfactants, as essentially amphiphilic molecules, show strong interactions or integration into lipid bilayers. Platelets (thrombocytes) respond to vascular injury by aggregating into thrombi. This process is determined mainly by membrane interactions that can be hindered by the presence of surfactants. Inhibition of this aggregation process, which can lead to hemostatic disorders, can be tested in vitro by activating platelets (from whole blood of healthy donors) with specific agonists, incubating them with the test sample and analyzing the degree of platelet inhibition. The aim of this experiment was therefore to test the degree of platelet inhibition caused by amphiphilic surfactants.

[0310] 23.2 Sample preparation: Human whole blood from six healthy volunteers was incubated with 0.04% or 0.01% surfactant for 10 min at 37°C. Saline solution was used as a negative control. Platelets were activated with four independent agonists: arachidonic acid (AA), thrombin receptor activator peptide (TRAP6), collagen and ADP. Aggregation was measured by whole blood aggregometry (WBA).

[0311] 23.3 Method: Whole blood aggregometry (WBA) consists of submerging electrodes in whole blood and measuring the impedance between the electrodes. Addition of a platelet agonist activates aggregation, resulting in an increase in impedance over time. This increase is compared between the negative control and the test sample to derive the degree of platelet inhibition.

[0312] 23.4 Results: As shown in Figure 5A, amphiphilic surfactants containing vitamin E, VEDS and VEDG 3.3 show platelet inhibition values ​​comparable to PS80, regardless of the agonist used. As shown in Figure 5B, SPGS-550-M has no effect on platelet aggregation. As shown in Figure 5C, while Brij58 and Brij58-succinate generally show higher inhibition of aggregation compared to PS80, the amount of inhibition is still not related to hemostatic impairment. Furthermore, in three out of four agonists shown in Figure 5C, Brij58-succinate shows lower platelet inhibition compared to Brij58, indicating a beneficial effect on the safety profile caused by the addition of a linker.

[0313] Example 24: Evaluation of detergent propensity for enzymatic degradation by four protein formulations (host cell proteins (HCPs)) 24.1 Purpose: Polysorbate 20 / 80, as mentioned above, suffers from enzymatic degradation mechanism.To test the amphiphilic surfactant of the present disclosure for stability against enzymatic degradation, the following assay was carried out.The purpose was to test whether amphiphilic surfactant is susceptible to the same enzymatic degradation that is effective in PS80.

[0314] 24.2 Sample preparation: Protein 4 is a monoclonal antibody whose production process resulted in contaminant HPC enzymes, a common situation as described above. Therefore, formulations containing protein 4 also contain enzymes involved in polysorbate degradation, which are suitable proxies for testing the propensity for enzyme degradation during shelf life in real-world conditions. The formulations were supplemented with either 0.02% PS80 or 0.04% amphiphilic surfactants VEDS, VEDG 2.2 or TPGS-1000 and incubated at either 5°C or 40°C for one month.

[0315] 24.3 Method: Surfactant content was measured by HPLC at the required pull points and analysis was performed by standard baseline correction followed by integration of the main peak representing the integral (unresolved) surfactant.

[0316] 24.4 Results: As shown in Figure 6, enzymatic degradation originating from protein 4 host cell proteins (HCPs) significantly affects PS80. Degradation is observed at both 5°C and 40°C, with stronger effects predicted at higher temperatures. VEDS, VEDG 2.2 and TPGS-1000 are not affected by enzymatic degradation after one month, regardless of incubation temperature.

[0317] Example 25: Evaluation of surfactant propensity for oxidation and hydrolysis in static stability studies 25.1 Purpose: Polysorbate 20 / 80, as mentioned above, suffers from chemical degradation mechanisms. Stability studies were conducted to test the amphiphilic surfactants of the present disclosure for stability against oxidation and hydrolysis in long-term studies. The purpose was to test whether and, if so, to what extent the amphiphilic surfactants are susceptible to chemical degradation that affects PS80.

[0318] 25.2 Sample preparation: In the reported formulations, protein 2 was supplemented with either 0.04% PS80, VEDS, VEDG 2.2, TPGS-1000 or 0.01% Brij58 or SPGS-550-M and incubated for 6 months at 25° C. (FIG. 7). Pull points were taken at 1.5 month intervals (T0, T1.5m, T3m, T4.5m and T6m) and frozen for storage.

[0319] 25.3 Method: Surfactant content was measured for all pull points of a particular surfactant in a single series by HPLC and analysis was performed by standard baseline correction followed by integration of the main peak representing the integral (unresolved) surfactant.

[0320] 25.4 Results: As shown in Figure 7, chemical degradation significantly affected PS80 in this long-term study, with only a small amount of surfactant (approximately 5%) remaining after 6 months. VEDS, TPGS-1000, and Brij58 are clearly not susceptible to the same degradation, indicating a stable level of surfactant content. VEDG 2.2 has the least susceptibility to chemical degradation, indicating a stable level of surfactant content of approximately 88-90% after an initial drop in surfactant content. SPGS-550-M is susceptible to chemical degradation, which shows an approximately linear degradation, but is not as severe as in the case of PS80.

[0321] Example 26: Comparison of Therapeutic Protein (TP) Stabilization Effects Provided by Currently Used Surfactants to VEDS 26.1 Purpose: Comparison of therapeutic protein (TP) stabilization effects provided by currently used surfactants versus VEDS

[0322] 26.2 Sample preparation: Liquid formulations containing protein 5 were prepared as described herein, supplemented with either 0.2% PLX188 or 0.04% PS20, PS80 or VEDS, and incubated at 30° C. for one month ( FIG. 8 ). Pull points were then taken and used for the following analysis.

[0323] 26.3 Method: Protein purity was assessed by SEC-UPLC with standard baseline correction followed by integration of the main peak and identified by comparison with authentic samples, while the sum of impurities was calculated by integration of secondary peaks.

[0324] 26.4 Results: As shown in Figures 8A and 8B, all surfactants tested showed sufficient levels of API stabilization (>90% purity), while relative comparison between different surfactants results in rankings that may affect results in longer-term studies (i.e., 12 months). PS20 has lower purity and higher impurities. PLX188, PS80 and VEDS retain similar levels of purity and impurities, highlighting that the amphiphilic surfactant VEDS has a completely promising stabilizing effect.

[0325] summary The data described herein show that amphiphilic surfactants show less surfactant degradation during oxidation and hydrolysis stress compared to polysorbate 20 / 80. Vitamin E analogs, poly(alkylene glycol) alkyl ether analogs, and sitosterol analogs do not contain fatty acids as building blocks, and therefore, in addition to less peroxide generation during surfactant degradation, the risk of protein degradation leading to visible or non-visible particles is reduced. Improved stabilization effect is shown for those amphiphilic surfactants formed that contain dicarboxylic acid linkers, especially branched dicarboxylic acid alkyl linkers.

[0326] Finally, in vitro safety data suggests that the amphiphilic surfactants of the present disclosure do not cause adverse effects in primary human tissues with respect to hemolysis, B cell, or dendritic cell activation, and are therefore suitable as excipients for parenteral formulations.

[0327] Thus, the inventors have demonstrated the novel discovery of amphiphilic surfactants as stabilizers of protein APIs in liquid formulations and have developed novel surfactants of formula (I), (II) and (III) with predictable surfactant properties, physiological tolerability and with no to minimal drawbacks compared to the known drawbacks of polysorbates, particularly with regard to excipient instability.

Claims

1. A protein-containing formulation comprising a buffer and an amphiphilic surfactant, wherein the amphiphilic surfactant is represented by formula (I): 【Chemical 1】 (In the formula: Regarding formula (I): L １ is —C(═O)—X—C(═O)—; X is a branched chain C ２ ~C ２０ alkylene; R １ But Poly (C １ ~C ４ alkylene glycol) group, e.g., a PEG group, and optionally wherein said poly(C １ ~C ４ alkylene glycol) group is hydroxyl or C １ ~C ６ alkoxy terminated; R ２ , R ３ , R ４ and R ５ are each independently hydrogen and C １ ~C １０ alkyl; R ６ But C ５ ~C ２０ A protein-containing formulation, wherein the compound is a compound represented by the formula (I), wherein the compound is alkyl.

2. 10. The formulation of claim 1, wherein the amphiphilic surfactant is an amphiphilic antioxidant surfactant.

3. The formulation of claim 1 , wherein the protein is a disordered protein or an antibody.

4. The formulation of claim 3, wherein the antibody is an antibody-drug conjugate, a monoclonal antibody, or an antigen-binding fragment.

5. 4. The formulation of claim 3, wherein the antibody is present in an amount of 1 to 250 mg / ml.

6. The formulation of any one of claims 1 to 5, wherein the compound of formula (I) comprises a poly(alkylene glycol) moiety as the hydrophilic portion of the surfactant molecule.

7. The amphiphilic surfactant is represented by formula (I) １ is a PEG group, and the PEG group is hydroxyl or C １ ~C ６ The formulation according to any one of claims 1 to 5, wherein the compound is represented by the formula (I) wherein the compound is alkoxyl-terminated.

8. The formulation of claim 1, which is for subcutaneous administration.

9. 10. Use of an amphiphilic surfactant as a stabilizer for a formulation containing a protein, wherein the amphiphilic surfactant is a compound of formula (I) as defined in accordance with claim 1.

10. 10. Use of an amphiphilic surfactant as a stabilizer for a formulation containing a protein, wherein the amphiphilic surfactant is a compound of formula (I) as defined in accordance with claim 7.

11. The use according to claim 9, wherein the protein is defined according to any one of claims 3 to 5.

12. The use according to claim 10, wherein the protein is defined according to any one of claims 3 to 5.

13. Formula (I): 【Chemistry 2】 (For formula (I), wherein: L １ is —C(═O)—X—C(═O)—; X is a branched chain C ２ ~C ２０ alkylene; R １ But Poly (C １ ~C ４ alkylene glycol) groups, for example, PEG groups; R ２ , R ３ , R ４ and R ５ are each independently hydrogen and C １ ~C １０ alkyl; R ６ But C ５ ~C ２０ alkyl) surfactant.

14. The amphiphilic surfactant is represented by formula (I) １ is a PEG group, and the PEG group is hydroxyl or C １ ~C ６ 14. The surfactant according to claim 13, wherein the compound is a compound represented by the formula:

15. 14. A method for preventing or inhibiting in vitro protein aggregate formation in a protein-containing formulation by using an amphiphilic surfactant as defined in claim 13 as a stabilizer in said formulation.

16. 15. A method for preventing or inhibiting in vitro protein aggregate formation in a protein-containing formulation by using an amphiphilic surfactant as defined in claim 14 as a stabilizer in said formulation.

17. The method of claim 15, wherein the protein is defined according to any one of claims 3 to 5.

18. The method of claim 16, wherein the protein is defined according to any one of claims 3 to 5.

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