Preparations containing polysorbate

Abstract

Disclosed are formulations comprising polysorbate and citrate. Also disclosed are the use of citrate to prevent the degradation of polysorbate in aqueous formulations, and methods for preparing compositions, for example, compositions comprising peptides / polypeptides for use in therapy, prevention, and / or diagnosis.

Description

[Technical Field]

[0001] This application claims priority to European Patent No. 23163573.1, filed on 22 March 2023, the contents and elements of which are incorporated herein by reference for all purposes. This disclosure relates to pharmaceuticals, and more specifically, to biopharmaceutical formulation technology. [Background technology]

[0002] The development of biopharmaceutical products requires the evaluation of conditions and factors that may potentially affect product quality. Deterministic factors affecting protein stability include, for example, mechanical stress, temperature, exposure, freeze-thaw cycles, raw material impurities, and leaching from primary packaging (1-6). Based on these amphiphilic and physicochemical properties, proteins, such as monoclonal antibodies (mAbs), tend to adsorb to hydrophobic interfaces (e.g., solution / air interfaces, plastic polymers, glass, stainless steel), which may then partially denature, aggregate, and form particles (6, 7). A common goal in biopharmaceutical formulation development is to minimize such risks and ensure the safety and efficacy of the final drug product. Therefore, it is crucial to focus on the chemical, colloidal, and steric stability of proteins during drug development. The goal of drug development is to provide appropriate and patient-friendly formulations (e.g., buffer systems, isotonic agents, surfactants) for stabilizing proteins (e.g., mAbs). In addition, clinical operation and in-use stability testing is performed to examine the drug product, for example, using clinical dilution media and infusion media in combination. Because patient safety is paramount, such testing investigates protein stability and the possibility of protein particle formation. Especially for drugs delivered by infusion, dilution of the drug using infusion bags can reduce drug safety under dilution conditions (8-10).

[0003] Commercially available diluents used in clinics currently include physiological saline (0.9% (w / v) sodium chloride), 1 / 2 physiological saline, Ringer's lactate solution, and 5% (w / v) dextrose solution. Various research papers have reported incompatibility between proteins and such diluents and injection components (11-15). Particle formation phenomena have been described in the literature mainly in relation to highly diluted antibodies (e.g., monoclonal antibodies) (9, 14, 16-19). Some reports have evaluated the use of in-line filters to remove particles. However, particle removal requires validation of each in-line filter and product because the use of in-line filters may potentially affect the physicochemical stability of proteins and drug recovery (10, 11, 20). In certain cases, prescription information for diluting certain biologics, such as Herceptin® and Avastin®, requires the use of saline solution for drug dilution and specifically prohibits the use of dextrose ("Dextrose (5%) solution shall not be used") (15). Dilution with dextrose-containing solutions carries the risk of glycation between the sugar and the pharmaceutically active protein, in addition to the risk of affecting the physicochemical stability of the protein, which reduces efficacy by decreasing binding activity (21).

[0004] As a promising solution, a dedicated diluent having the same excipient composition as the drug product can be used. However, the use of a specific diluent for the drug product reduces flexibility in the clinic, necessitates further manufacturing processes and associated stability activities, and doubles the workload of the supply chain. To reduce the effort required to develop a suitable diluent, the existence of a general diluent that allows for the stabilization of most drug products in diluted applications is beneficial. Such diluents are useful for use in the preparation of infusions for use in clinics. For example, in a dose-escalation study in a Phase 1 cancer clinical trial, a highly diluted infusion was used, for example, 10 times the concentration of the drug product. 3 ~10 6 The drug product needs to be diluted to twice its original volume (22). Polysorbate (PS) is an amphiphilic nonionic surfactant with low toxicity, high biocompatibility, and high stabilization ability, and is therefore widely used in protein therapeutic formulations (3). Because PS is amphiphilic, it enhances the physical and colloidal stability of proteins by saturating hydrophobic interfaces (6, 23).

[0005] Polysorbate (PS) is composed of sorbitan / isosorbide bonded to polyoxyethylene (POE) chains via esterified fatty acids. The heterogeneity of PS is based on the degree of ethoxylation and esterification, as well as the mixture of fatty acids used in the preparation of PS. Polysorbate 20 (PS20) mainly contains esterified lauric acid (40-60%), while polysorbate 80 (PS80) mainly contains esterified oleic acid (>58%) (17, 24). The exact composition of official grade PS20 / PS80 is defined in the pharmacopoeia (Ph.Eur., USP, JP, BP or ChP). PS20 and PS80 are also known as Tween® 20 and Tween® 80, respectively. Figure 1 shows the ideal chemical structure of PS20. The stability of PS in aqueous formulations exhibits certain tendencies. Two main degradation pathways are currently considered: hydrolysis pathways (chemical and enzymatic hydrolysis) targeting fatty acid ester bonds and oxidation pathways targeting hydrocarbon chain double bonds and / or polyoxyethylene (POE) chains of unsaturated fatty acids (25, 26) (Figure 1). Both degradation pathways pose a potential risk to the integrity and shelf life of the active pharmaceutical ingredient (27, 28) and to clinically important diluents containing PS.

[0006] While host cell proteins have been identified as the primary cause of hydrolytic enzymatic degradation (29, 30), several factors can promote the oxidative degradation of PS. In principle, the presence of reactive oxygen species (ROS) is essential (31, 32). Since H2O2 is used as a decontamination agent during manufacturing, the source of ROS, such as residual hydrogen peroxide (H2O2) or organic peroxides, can be the raw materials or the manufacturing / filling process (33, 34). Such factors degrade PS, and consequently, the surfactant functionality of PS is impaired, leading to the instability of the drug product contained in the formulation (35). Current biopharmaceutical research primarily concerns the hydrolysis of PS20 in biopharmaceutical formulations due to the presence of enzymes (36, 37). Various strategies to mitigate the degradation of PS have been investigated. For example, the effects of various excipients on the oxidative degradation of PS, such as EDTA, methionine, citrate, or butylhydroxytoluene (BHT), have been evaluated, but a wide range of conclusions have been reported, and dual effects have even been observed (24, 38-40). Doyle et al. (38) state that PS80 remains stable for several days in high buffer concentrations of 10 mM histidine buffer or 10 mM citrate buffer. Doyle et al. also report that histidine buffer containing citrate prevents oxidation of PS80 for up to 7 days in short-term tests. Gopalrathnam et al. (47) describe the degradation tests of PS80 and PS20 with high concentrations of histidine and / or citrate, specifically with a histidine buffer and / or a 10 mM citrate buffer. [Overview of the Initiative]

[0007] In a first embodiment, the present disclosure provides an aqueous formulation comprising a polysorbate, a citrate, and sodium chloride. The present disclosure also provides an aqueous formulation essentially comprising a polysorbate, a citrate, sodium chloride, and water. In some embodiments, the polysorbate is polysorbate 80. Preferably, the polysorbate is polysorbate 20. In particular, this disclosure provides an aqueous formulation comprising polysorbate 20, citrate, and sodium chloride. Furthermore, this disclosure provides an aqueous formulation essentially consisting of polysorbate 20, citrate, sodium chloride, and water. In some embodiments of various aspects of this disclosure, the concentration of citrate in the aqueous formulation is less than 10 mM, preferably less than 5 mM, and more preferably less than 3 mM. If the citrate concentration is high, for example, when a protein or peptide is added, protein particles may be formed and / or gelation may be promoted. Therefore, the concentration of citrate in the aqueous formulation is preferably about 50 μM to about 2.5 mM, more preferably about 250 μM to about 1 mM. Even more preferably, the concentration of citrate in the aqueous formulation is about 0.25 mM to about 0.75 mM. Most preferably, the concentration of citrate is about 0.5 mM.

[0008] In some embodiments, the concentration of polysorbate (e.g., polysorbate 20 or polysorbate 80) in the aqueous formulation is approximately 0.02% (w / v). Furthermore, this disclosure provides the use of citrate to prevent the degradation of polysorbate in an aqueous formulation, wherein the final molar ratio of citrate to polysorbate is about 1.5 to about 7. It also provides a method for preventing the degradation of polysorbate in an aqueous formulation, comprising the step of adding citrate in a final molar ratio of citrate to polysorbate of about 1.5 to about 7. In some embodiments, the polysorbate is polysorbate 80. Preferably, the polysorbate is polysorbate 20. In the aqueous formulations, methods, or uses of the present disclosure, the molar ratio (or final molar ratio) of citrate to polysorbate in the aqueous formulation is about 1.5 to about 7, preferably about 3 to about 6. More preferably, the molar ratio (or final molar ratio) of citrate to polysorbate is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9.

[0009] Furthermore, this disclosure provides the use of citrate to prevent the degradation of polysorbate 20 in an aqueous formulation, wherein the final molar ratio of citrate to polysorbate 20 is about 0.31 to about 15.4, preferably about 1.5 to about 7. Also provided is a method for preventing the degradation of polysorbate 20 in an aqueous formulation, comprising the step of adding citrate, wherein the final molar ratio of citrate to polysorbate is about 1.5 to about 7. In the aqueous formulations, methods, or uses of the present disclosure, the molar ratio (or final molar ratio) of citrate to polysorbate 20 in the aqueous formulation is preferably about 1.5 to about 7. More preferably, the molar ratio (or final molar ratio) of citrate to polysorbate 20 is about 3 to about 6. In certain embodiments, the molar ratio (or final molar ratio) of citrate to polysorbate 20 is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In the aqueous formulations, methods, or uses of the present disclosure, the molar ratio (or final molar ratio) of citrate to polysorbate 80 in the aqueous formulation is preferably about 1.6 to about 7. More preferably, the molar ratio (or final molar ratio) of citrate to polysorbate 80 is about 3.2 to about 6.5. In certain embodiments, the molar ratio (or final molar ratio) of citrate to polysorbate 80 is about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9.

[0010] In some embodiments of the aqueous formulations, methods or uses of the present disclosure, the aqueous formulation comprises sodium chloride at a concentration of about 0.9% (w / v). In some embodiments, the aqueous formulation does not contain histidine and / or further contains metal ions. In some embodiments, the concentration of sodium chloride in the aqueous formulation is about 0.9% (w / v), and the molar ratio of citrate to polysorbate (e.g., polysorbate 20, polysorbate 80, etc.) in the aqueous formulation is about 3 to about 6. In some embodiments, the aqueous formulation consists essentially of polysorbate 20, citrate, water, and sodium chloride. In some embodiments, the aqueous formulation further comprises a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. In some embodiments, the aqueous formulation further comprises a peptide / polypeptide or a complex thereof for use in therapy.

[0011] In some embodiments of the aqueous formulations, methods or uses of the present disclosure, the aqueous formulation is - about 0.02% (w / v) polysorbate, - about 0.9% (w / v) sodium chloride, - water, - 0.25 mM to 1 mM citrate, and - optionally, a pharmaceutically acceptable carrier, excipient or adjuvant, - optionally, a peptide / polypeptide for use in therapy, prophylaxis, and / or diagnosis and consists essentially of these. Preferably, this aqueous formulation is - about 0.02% (w / v) polysorbate, - about 0.9% (w / v) sodium chloride, - water, - about 0.5 mM citrate, and - optionally, a pharmaceutically acceptable carrier, excipient or adjuvant, - optionally, a peptide / polypeptide for use in therapy, prophylaxis, and / or diagnosis and consists essentially of these.

[0012] In some embodiments of the aqueous formulations, methods, or uses of the present disclosure, the aqueous formulation comprises polysorbate 80. - Approximately 0.02% (w / v) of polysorbate 80, - Approximately 0.9% (w / v) sodium chloride, - water, - 0.25 mM to 1 mM citrate, and - Optionally, a pharmaceutically acceptable carrier, excipient, or adjuvant. - Optionally, peptides / polypeptides for use in therapy, prevention, and / or diagnosis. It becomes essentially. Preferably, this aqueous formulation containing polysorbate 80, - Approximately 0.02% (w / v) of polysorbate 80, - Approximately 0.9% (w / v) sodium chloride, - water, - Approximately 0.5 mM citrate, and - Optionally, a pharmaceutically acceptable carrier, excipient, or adjuvant. - Optionally, peptides / polypeptides for use in therapy, prevention, and / or diagnosis. It becomes essentially.

[0013] In other embodiments of the aqueous formulations, methods, or uses of the present disclosure, the aqueous formulation comprises polysorbate 20. - Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - water, - 0.25 mM to 1 mM citrate, and - Optionally, a pharmaceutically acceptable carrier, excipient, or adjuvant. - Optionally, peptides / polypeptides for use in therapy, prevention, and / or diagnosis. It becomes essentially. Preferably, this aqueous formulation containing polysorbate 20, - Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - water, - Approximately 0.5 mM citrate, and - Optionally, a pharmaceutically acceptable carrier, excipient, or adjuvant. - Optionally, peptides / polypeptides for use in therapy, prevention, and / or diagnosis. It becomes essentially.

[0014] In some embodiments, aqueous formulations are suitable for intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. Furthermore, this disclosure provides the use of any of the aqueous formulations described herein as a diluent for the composition. Furthermore, the Disclosure provides a method for preparing a composition, comprising the step of contacting a peptide / polypeptide or a complex thereof for use in therapy, prevention, and / or diagnosis with an aqueous formulation according to the Disclosure. In some embodiments, the method is a method for preparing a pharmaceutical composition for administration.

[0015] In some embodiments, peptides / polypeptides or complexes thereof for use in therapy, prevention, and / or diagnosis are selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. Furthermore, this disclosure provides compositions that can be obtained or obtained by any of the methods of this disclosure. Here, embodiments and experiments illustrating the principles of this disclosure will be discussed with reference to the attached drawings. [Brief explanation of the drawing]

[0016] [Figure 1]This diagram shows the ideal chemical structure of PS20 (POE sorbitan monoester) and the possible sites of oxidative decomposition (dark gray) and hydrolysis (light gray). W+x+y+z=20, which refers to the number of ethylene oxide moieties as defined by the pharmacopoeia. [Figure 2] This figure shows that a titration series of citrate-to-polysorbate 20 molar ratios from 0 to 15 defined the optimal range for mitigating the oxidation of PS20. Formulations of 0.2 mg / ml-1 PS20 in 0.9% (w / v) NaCl at different citrate-to-PS20 molar ratios are shown. Oxidative stress stability tests were performed at 40°C / 75% relative humidity (rH) in the presence of 29 mM H2O2. PS20 content was determined by fluorescence micelle assay (FMA). All values ​​represent a technical octuplet (n=8) of two replicates. The mean and standard deviation (SD) values ​​of the replicates are reported. [Figure 3A] This figure shows the reduction of oxidative degradation of PS20 in diluted formulations during long-term stability testing. It shows formulations of 0.2 mg / ml-1 PS20 in MilliQ water or 0.9% (w / v) NaCl in the presence and absence of citrate. The citrate-to-PS20 molar ratio was 3.07. The figures show samples from oxidative long-term stability tests conducted at 40°C / 75% rH for 12 months (Figure 3A) and up to 24 months (Figure 3B) in the presence of 29 μM H2O2. PS20 content was determined by FMA. The figures show PS20 formulations in MilliQ water (gray bars) or 0.9% (w / v) NaCl (white bars) in the absence of citrate. The figures also show PS20 formulations in MilliQ water (gray dotted bars) or 0.9% (w / v) NaCl (white dotted bars) in the presence of citrate. All values ​​represent a technical octuplet (n=8) from two replicates. Report the mean and standard deviation (SD) values ​​of the repeated measurements. [Figure 3B] Same as above. [Figure 4]This figure shows the oxidation markers of oxidative degradation of PS20 in diluted formulations. The figures show PS20 formulations of 0.2 mg / ml-1 in 0.9% (w / v) NaCl in the absence (A) and presence (B) of citrate. The citrate-to-PS20 molar ratio was 3.07. The figures also show samples from long-term oxidative stability tests conducted in the presence of 29 μM H2O2 and stored at 40°C / 75% rH for up to 12 months. The content of oxidation markers (POE5 Di, POE5 Mono, POE6 Di, POE6 Mono) was determined by UPLC-QDA assay. (A) Initial and 3-month PS20 formulations in the absence of citrate. (B) Initial and 6-month PS20 formulations in the presence of citrate. [Figure 5] This figure shows that a titration series of citrate-to-polysorbate 80 molar ratios from 0 to 16 defined the optimal range for mitigating the oxidation of PS80. Formulations of 0.2 mg / ml-1 PS80 in 0.9% (w / v) NaCl at different citrate-to-PS80 molar ratios are shown. Oxidative stress stability tests were performed at 40°C / 75% relative humidity (rH) in the presence of 29 mM H2O2. PS80 content was determined by fluorescence micelle assay (FMA). All values ​​represent a technical octuplet (n=8) of two replicates. The mean and standard deviation (SD) values ​​of the replicates are reported. [Figure 6A] This figure shows the turbidity measurements of mAb 1 formulations (D00-D005) in the absence (6A) and presence (6B) of citrate. The solutions were stored either in the dark or under exposure. Turbidity was measured in formazin turbidimetric units (FNU) using a Hach-Lange instrument at wavelengths of 400-600 nm at the following time points: 0 hours, 9 hours, and 24 hours. [Figure 6B] Same as above [Figure 7] This table shows the results of visual inspection of mAb 1 formulations (D00-D005) conducted according to the European Pharmacopoeia (Ph.Eur. 2.9.20). The solutions were stored in either the dark or under light, and evaluated by visual inspection at the following time points: 0 hours, 9 hours, and 24 hours. [Figure 8A]This figure shows the concentrations of microscopic particles with a size of ≥10 μm in each mAb 1 formulation (D00~D005) in the absence (8A) and presence (8B) of citrate, as determined by microflow imaging (MFI). [Figure 8B] Same as above [Figure 9A] This figure shows the concentration of microscopic particles with a size of ≥25 μm in each mAb 1 formulation (D00~D005) in the absence (9A) and presence (9B) of citrate, as determined by microflow imaging (MFI). [Figure 9B] Same as above [Modes for carrying out the invention]

[0017] This disclosure relates to aqueous formulations containing polysorbate and citrate, and more particularly to aqueous formulations containing polysorbate 20 and citrate. In the experimental examples of this disclosure, the inventors demonstrate that such formulations stabilize (i.e., prevent the degradation of) polysorbates, such as polysorbate 20 and polysorbate 80.

[0018] Using a citrate to polysorbate (e.g., polysorbate 80 or polysorbate 20) molar ratio within an optimal range of approximately 1.5 to approximately 7, preferably approximately 1.6 to approximately 7, and more preferably approximately 3 to approximately 6, has been shown to prevent polysorbate degradation. Low ratios (e.g., less than approximately 1.6) or high ratios (e.g., approximately 16) have been shown to be less effective in preventing the corresponding polysorbate degradation in aqueous formulations. In particular, it has been demonstrated that using a citrate-to-polysorbate 20 molar ratio within an optimal range of about 1.5 to about 7, preferably about 3 to about 6, prevents the degradation of polysorbate 20. It has been demonstrated that low ratios (e.g., less than about 1.5) or high ratios (e.g., about 15) are less effective in preventing the degradation of PS20 in aqueous formulations.

[0019] Polysorbate Polysorbates (PS) are a type of amphiphilic nonionic surfactant. The term "polysorbate" refers to surfactants composed of sorbitan / isosorbide bonded to a polyoxyethylene (POE) chain via esterified fatty acids. Examples of polysorbates include, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80. Polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 utilize lauric acid, palmitic acid, stearic acid, and oleic acid, respectively, in the fatty acid portion of their molecules. Polysorbate 20 (PS20) is polyoxyethylene sorbitan monolaurate. Polysorbate 80 (PS80) is polyoxyethylene sorbitan monooleate. Polysorbate 20 (PS20) mainly contains esterified lauric acid (40-60%), while polysorbate 80 (PS80) mainly contains esterified oleic acid (>58%) (17, 24). The exact composition of official grade PS20 / PS80 is defined in the pharmacopoeia (Ph.Eur., USP, JP, BP or ChP). Polysorbate 40 is polyoxyethylene sorbitan monopalmitate. Polysorbate 60 is polyoxyethylene sorbitan monostearate, while polysorbate 65 is polyoxyethylene sorbitan tristearate.

[0020] In some embodiments, the polysorbate is or comprises polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80. In some embodiments of this disclosure, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the polysorbate is polysorbate 60. In some embodiments, the polysorbate is polysorbate 40. Preferably, in this disclosure, the polysorbate is polysorbate 20.

[0021] In some embodiments, the concentration of polysorbate (e.g., polysorbate 80) in the aqueous formulation according to this disclosure is approximately 0.001% (w / v) (=0.01 g / L) to approximately 0.06% (w / v) (=0.6 g / L), for example, approximately 0.001% (w / v) to approximately 0.01% (w / v), approximately 0.005% to approximately 0.01% (w / v), approximately 0.005% (w / v) to approximately 0.015%, approximately 0.01% (w / v) to approximately 0.02%, approximately 0.015% (w / v) to approximately 0.02% (w / v), approximately 0.015% (w / v) to approximately 0.025% (w / v), approximately 0.02% (w / v) to approximately 0.03% (w / v), and approximately 0.025% (w / v) ~ approx. 0.03%, approx. 0.025% (w / v) ~ approx. 0.035% (w / v), approx. 0.03% (w / v) ~ approx. 0.04% ( w / v), approx. 0.035% (w / v) ~ approx. 0.04% (w / v), approx. 0.035% (w / v) ~ approx. 0.045% (w / v), approx. 0.04% (w / v) is one of the following concentrations: approximately 0.05% (w / v), approximately 0.045% (w / v) to approximately 0.05% (w / v), approximately 0.045% (w / v) to approximately 0.055% (w / v), approximately 0.05% (w / v) to approximately 0.06% (w / v), and approximately 0.055% (w / v) to approximately 0.06% (w / v). In some embodiments, the concentration is one of the following: approximately 0.01%(w / v) to approximately 0.06%(w / v), for example, approximately 0.02% to approximately 0.06%(w / v), approximately 0.03%(w / v) to approximately 0.06%(w / v), approximately 0.04%(w / v) to approximately 0.06%(w / v), and approximately 0.05%(w / v) to approximately 0.06%(w / v).

[0022] The concentration of polysorbate (e.g., polysorbate 80) in the aqueous formulation according to this disclosure is preferably about 0.01% (w / v) to about 0.06% (w / v), preferably about 0.015% (w / v) to about 0.04% (w / v), 0.02% (w / v) to about 0.04% (w / v), more preferably about 0.015% (w / v) to about 0.03% (w / v), 0.02% (w / v) to about 0.03% (w / v), and even more preferably about 0.02% (w / v). A concentration of 0.02% (w / v) corresponds to 0.2 mg / ml -1 It is equivalent to. In other embodiments, the concentration of polysorbate (e.g., polysorbate 80) is about 0.01% (w / v). It is understood that higher concentrations of polysorbate may be used. Therefore, in some embodiments, the concentration of polysorbate (e.g., polysorbate 80) in the aqueous formulation is about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), or about 0.06% (w / v).

[0023] In some embodiments, the concentration of polysorbate (e.g., polysorbate 80) in the aqueous formulation according to this disclosure is approximately 8 μM to approximately 0.46 mM, for example, approximately 8 μM to approximately 0.08 mM, approximately 0.04 mM to approximately 0.08 mM, approximately 0.04 mM to approximately 0.11 mM, approximately 0.08 mM to approximately 0.14 mM, approximately 0.10 mM to approximately 0.15 mM, approximately 0.08 mM to approximately 0.19 mM, and approximately 0.17 mM to approximately 0.23 mM. It is one of the following concentrations: approximately 0.19 mM to approximately 0.23 mM, approximately 0.19 mM to approximately 0.23 mM, approximately 0.23 mM to approximately 0.31 mM, approximately 0.27 mM to approximately 0.31 mM, approximately 0.27 mM to approximately 0.34 mM, approximately 0.31 mM to approximately 0.38 mM, approximately 0.34 mM to approximately 0.38 mM, approximately 0.34 mM to approximately 0.42 mM, approximately 0.38 mM to approximately 0.46 mM, and approximately 0.42 mM to approximately 0.46 mM.

[0024] The concentration of polysorbate (e.g., polysorbate 80) in the aqueous formulation according to this disclosure is preferably about 80 μM to about 0.49 mM, preferably about 0.12 mM to about 0.33 mM, 0.17 mM to about 0.33 mM, more preferably about 0.12 mM to about 0.25 mM, 0.15 mM to about 0.25 mM, and even more preferably about 0.15 mM. For example, a PS80 concentration of 0.15 mM is 0.2 mg / ml -1 It is equivalent to. In other embodiments, the concentration of polysorbate (e.g., polysorbate 80) is about 0.008 mM. It is understood that higher concentrations of polysorbate may be used. Therefore, in some embodiments, the concentration of polysorbate in the aqueous formulation is about 0.008 mM, about 0.15 mM, about 0.23 mM, about 0.31 mM, about 0.38 mM, or about 0.46 mM. In some embodiments of various aspects of this disclosure, the polysorbate is not polysorbate 80 (PS80).

[0025] Preferably, in this disclosure, polysorbate is polysorbate 20. In some embodiments, the concentration of polysorbate 20 in the aqueous formulation according to this disclosure is approximately 0.001% (w / v) (=0.01 g / L) to approximately 0.06% (w / v) (=0.6 g / L), for example, approximately 0.001% (w / v) to approximately 0.01% (w / v), approximately 0.005% to approximately 0.01% (w / v), approximately 0.005% (w / v) to approximately 0.015%, approximately 0.01% (w / v) to approximately 0.02%, approximately 0.015% (w / v) to approximately 0.02% (w / v), approximately 0.015% (w / v) to approximately 0.025% (w / v), approximately 0.02% (w / v) to approximately 0.03% (w / v), and approximately 0.025%. This represents one of the following concentrations: (w / v) ~ approximately 0.03%, approximately 0.025%(w / v) ~ approximately 0.035%(w / v), approximately 0.035%(w / v) ~ approximately 0.04%(w / v), approximately 0.035%(w / v) ~ approximately 0.045%(w / v), approximately 0.04%(w / v) ~ approximately 0.05%(w / v), approximately 0.045%(w / v) ~ approximately 0.05%(w / v), approximately 0.045%(w / v) ~ approximately 0.055%(w / v), approximately 0.05%(w / v) ~ approximately 0.06%(w / v), and approximately 0.055%(w / v) ~ approximately 0.06%(w / v). In some embodiments, the concentration is one of the following: approximately 0.01%(w / v) to approximately 0.06%(w / v), for example, approximately 0.02% to approximately 0.06%(w / v), approximately 0.03%(w / v) to approximately 0.06%(w / v), approximately 0.04%(w / v) to approximately 0.06%(w / v), and approximately 0.05%(w / v) to approximately 0.06%(w / v). The concentration of polysorbate 20 in the aqueous formulation according to this disclosure is preferably about 0.01% (w / v) to about 0.06% (w / v), preferably about 0.015% (w / v) to about 0.04% (w / v), 0.02% (w / v) to about 0.04% (w / v), more preferably about 0.015% (w / v) to about 0.03% (w / v), 0.02% (w / v) to about 0.03% (w / v), and even more preferably about 0.02% (w / v). A concentration of 0.02% (w / v) corresponds to 0.2 mg / ml -1 It is equivalent to.

[0026] In other embodiments, the concentration of polysorbate 20 is about 0.01% (w / v). It is understood that higher concentrations of polysorbate 20 may be used. Therefore, in some embodiments, the concentration of polysorbate 20 in the aqueous formulation is about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), or about 0.06% (w / v). The concentration of polysorbate in the aqueous formulations according to this disclosure may be expressed in molar concentration (mol / L or M). Therefore, the (w / v) concentration can be converted to molar concentration using the molecular weight of the polysorbate. For the purposes of such calculations, the molecular weight of PS20 used is 1227 g / mol. For the purposes of such calculations, for example, the molecular weight of PS80 used for comparison is 1310 g / mol. In some embodiments, the concentration of polysorbate 20 in the aqueous formulation according to this disclosure is approximately 8 μM to approximately 0.48 mM, for example, approximately 8 μM to approximately 0.08 mM, approximately 0.04 mM to approximately 0.08 mM, approximately 0.04 mM to approximately 0.12 mM, approximately 0.08 mM to approximately 0.16 mM, approximately 0.12 mM to approximately 0.16 mM, approximately 0.08 mM to approximately 0.2 mM, approximately 0.18 mM to approximately 0.24 mM, and approximately 0.2 mM. M represents one of the following concentrations: approximately 0.24 mM, approximately 0.2 mM to approximately 0.28 mM, approximately 0.24 mM to approximately 0.32 mM, approximately 0.28 mM to approximately 0.32 mM, approximately 0.28 mM to approximately 0.36 mM, approximately 0.32 mM to approximately 0.41 mM, approximately 0.36 mM to approximately 0.41 mM, approximately 0.36 mM to approximately 0.45 mM, approximately 0.41 mM to approximately 0.48 mM, and approximately 0.45 mM to approximately 0.48 mM.

[0027] The concentration of polysorbate 20 in the aqueous formulation according to this disclosure is preferably about 80 μM to about 0.49 mM, preferably about 0.12 mM to about 0.33 mM, 0.17 mM to about 0.33 mM, more preferably about 0.12 mM to about 0.25 mM, 0.17 mM to about 0.25 mM, and even more preferably about 0.17 mM. A concentration of 0.17 mM corresponds to 0.2 mg / ml -1 It is equivalent to. In other embodiments, the concentration of polysorbate 20 is about 80 μM. It is understood that higher concentrations of polysorbate 20 may be used. Therefore, in some embodiments, the concentration of polysorbate 20 in the aqueous formulation is about 80 μM, about 0.17 mM, about 0.25 mM, about 0.33 mM, about 0.41 mM, or about 0.49 mM.

[0028] Aqueous formulation "Aqueous formulation" refers to a solution containing water. In preferred embodiments, the aqueous formulation according to this disclosure utilizes water as the solvent. The aqueous formulation according to this disclosure preferably comprises one or more solute compounds (i.e., one or more compounds dissolved in a solvent). In some embodiments, the primary solvent for the solute compounds in the aqueous formulation according to this disclosure is water. Furthermore, the aqueous formulations of this disclosure may be used as diluents. For example, the aqueous formulations may be used as diluents for substances of interest, such as substances useful in therapy, prevention, and / or diagnosis, or as diluents for compounds containing such substances of interest. Accordingly, the aqueous formulations of this disclosure may consist of or essentially consist of polysorbate 20, citrate, sodium chloride, and water. The aqueous formulations according to this disclosure may consist of or essentially consist of polysorbate (e.g., polysorbate 80), citrate, sodium chloride, and water.

[0029] As used herein, a composition (e.g., an aqueous solution) "consisting essentially of" or "consists essentially of" one or more specific components does not contain any further non-specific components in trace amounts or more. In some embodiments, in particular, in a composition (e.g., an aqueous solution) "consisting essentially of" one or more specific components, any further non-specific components constitute less than 5% (w / v) of the composition, for example, one of ≤1% (w / v), ≤0.5% (w / v), ≤0.1% (w / v), ≤0.05% (w / v), or ≤0.01% (w / v) of the composition. As an example, in an aqueous formulation according to this disclosure essentially consisting of polysorbate 20, citrate, sodium chloride, and water, any component of the aqueous formulation other than polysorbate 20, citrate, sodium chloride, and water constitutes less than 5% (w / v) of the composition, for example, one of ≤1% (w / v), ≤0.5% (w / v), ≤0.1% (w / v), ≤0.05% (w / v), or ≤0.01% (w / v) of the aqueous formulation. In some embodiments, any further non-specific component is present at a concentration of less than 5 mM, for example, one of ≤1 mM, ≤0.5 mM, ≤0.1 mM, ≤0.05 mM, or ≤0.01 mM. As an example, in an aqueous formulation according to this disclosure consisting essentially of polysorbate, citrate, sodium chloride, and water, any component of the aqueous formulation other than polysorbate, citrate, sodium chloride, and water has a concentration of less than 5 mM in the aqueous formulation, for example, one of ≤1 mM, ≤0.5 mM, ≤0.1 mM, ≤0.05 mM, or ≤0.01 mM. It is understood that a composition consisting essentially of one or more specific components encompasses a composition consisting of only one or more specific components. Therefore, in some embodiments, a composition consisting essentially of one or more specific components may be such a composition consisting of one or more specific components.

[0030] The aqueous formulation, as described herein, contains a polysorbate, such as polysorbate 20 or polysorbate 80. Preferably, the polysorbate contained in the aqueous formulation is polysorbate 20. In some embodiments, the aqueous formulation contains sodium chloride (NaCl). In some embodiments, the aqueous formulation contains physiological saline. Therefore, the aqueous formulation preferably contains sodium chloride at a concentration of about 0.9% (w / v) (=9 g / L). Therefore, in some embodiments, the aqueous formulation is an isotonic solution. In other embodiments, the aqueous formulation contains half saline solution. That is, the aqueous formulation contains 0.45% (w / v) sodium chloride. Therefore, the aqueous formulation is hypotonic in some embodiments.

[0031] In some embodiments, the concentration of NaCl in the aqueous formulation according to this disclosure is approximately 0.1% (w / v) (=g / L) to approximately 1.7% (w / v) (=17g / L), for example, approximately 0.1% (w / v) to approximately 1.7% (w / v), approximately 0.2% (w / v) to approximately 1.6% (w / v), approximately 0.3% (w / v) to approximately 1.5% (w / v), and approximately 0.5% The concentration is one of the following: (w / v) ~ approximately 1.4%(w / v), approximately 0.4%(w / v) ~ approximately 1.3%(w / v), approximately 0.5%(w / v) ~ approximately 1.2%(w / v), approximately 0.6%(w / v) ~ approximately 1.1%(w / v), approximately 0.7%(w / v) ~ approximately 1.0%(w / v), or approximately 0.85%(w / v) ~ approximately 0.95%(w / v). In some embodiments, the concentration of NaCl in the aqueous formulation according to this disclosure is approximately 0.9%(w / v). In some embodiments, the aqueous formulation includes Ringer's lactate solution.

[0032] In some embodiments, the aqueous formulation contains dextrose. In some embodiments, the concentration of dextrose in the aqueous formulation according to this disclosure is about 2.5% (w / v) to about 50% (w / v), for example, one of the following concentrations: about 2.5% (w / v) to about 5%, about 5% (w / v) to about 10% (w / v), about 10% (w / v) to about 20% (w / v), about 20% (w / v) to about 30% (w / v), about 30% (w / v) to about 40% (w / v), and about 40% (w / v) to about 50% (w / v). In some embodiments, the concentration of dextrose is about 2.5% (w / v), about 5% (w / v), about 10% (w / v), about 20% (w / v), about 30% (w / v), or about 50% (w / v). In some embodiments, the dextrose concentration is approximately 5% (w / v). In some embodiments, the aqueous formulation contains approximately 0.9% (w / v) sodium chloride and approximately 0.2 mg / ml -1 It contains a polysorbate (e.g., PS80). Preferably, the polysorbate is polysorbate 20. Therefore, in certain embodiments, the aqueous formulation contains about 0.9% (w / v) sodium chloride and about 0.2 mg / ml -1 Contains polysorbate 20 (PS20).

[0033] The aqueous formulations according to this disclosure contain citrates. Citrates are anions formed by the deprotonation of one or more carboxyl groups of the tricarboxylic acid that is citric acid. These can be obtained in solution by adding citric acid or a salt of citric acid (e.g., citric acid monohydrate) to water. For example, citrates in aqueous formulations can be obtained by contacting water with a salt of citric acid, such as sodium citrate dihydrate or trisodium citrate. Suitable salts of citric acid include sodium citrate dihydrate, disodium citrate, trisodium citrate, and combinations thereof. Preferably, sodium citrate (dihydrate) can be used to obtain the aqueous formulations according to this disclosure. This disclosure presents citrate-to-polysorbate molar ratios that are advantageously useful in preventing the degradation of polysorbate (e.g., PS80) in aqueous formulations. The aqueous formulations of this disclosure may include the following molar ratios. This disclosure also provides the use of citrate to prevent the degradation of polysorbate (e.g., PS80) in aqueous formulations described herein, wherein the final molar ratio of citrate to polysorbate (e.g., PS80) is as described herein.

[0034] This disclosure presents molar ratios of citrate to polysorbate 20 that are advantageously useful in preventing the degradation of polysorbate 20 in aqueous formulations. The aqueous formulations of this disclosure may include the following molar ratios. This disclosure also provides the use of citrate to prevent the degradation of polysorbate 20 in aqueous formulations described herein, wherein the final molar ratio of citrate to polysorbate 20 is as described herein. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 1.5 to about 7. In certain embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 1.6 to about 7. In certain embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3 to about 7. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 1.5 to about 6. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 1.6 to about 6. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3 to about 6. Preferably, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3.2 to about 6.5. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3 to about 5.

[0035] In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 3.3. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, or about 4.9. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, or about 5.9. In some embodiments, the molar ratio of citrate to polysorbate (e.g., PS80) in the aqueous formulation is about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9.

[0036] In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 1.5 to about 7. In certain embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3 to about 7. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 1.5 to about 6. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3 to about 6. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3 to about 5. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, or about 4.9.

[0037] In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, or about 5.9. In some embodiments, the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9. In some embodiments, the aqueous formulation contains a buffering agent. The buffering agent is used to maintain the pH of the aqueous formulation through the action of an acid-base conjugate component. In some embodiments, the buffering agent is not histidine. In some embodiments, the buffering agent is not a phosphate. In some embodiments, the aqueous formulation does not contain histidine. For example, the aqueous formulation does not contain histidine buffers. In some embodiments, the aqueous formulation does not contain histidine buffer salts; for example, the aqueous formulation does not contain any of histidine malate, histidine maleate, histidine fumarate, histidine tartrate, histidine phosphate, histidine lactate, histidine succinate, and / or histidine hydrochloride. In some embodiments, the aqueous formulation does not contain histidine hydrochloride. In some embodiments, the aqueous formulation does not contain histidine hydrochloride and histidine.

[0038] Trehalose, sucrose, mannitol, sorbitol, methionine, histidine, glycine, arginine, and combinations thereof are commonly used as stabilizers in pharmaceutical formulations, such as protein formulations. In some embodiments, the aqueous formulation does not contain sucrose. In some embodiments, the aqueous formulation does not contain trehalose, sucrose, mannitol, sorbitol, methionine, histidine, glycine, and / or arginine. In some embodiments, the aqueous formulation is phosphate-free; for example, the aqueous formulation is sodium phosphate and / or potassium phosphate-free. In some embodiments, the aqueous formulation is substantially histidine-free. For example, the aqueous formulation is substantially histidine buffer-free. In some embodiments, the aqueous formulation is substantially histidine buffer salt-free; for example, the aqueous formulation is substantially free of any of histidine malate, histidine maleate, histidine fumarate, histidine tartrate, histidine citrate, histidine phosphate, histidine lactate, histidine succinate, and / or histidine hydrochloride. In some embodiments, the aqueous formulation is substantially histidine hydrochloride-free. In some embodiments, the aqueous formulation is substantially histidine hydrochloride-free and histidine-free. In some embodiments, the aqueous formulation is substantially free of sucrose. In some embodiments, the aqueous formulation is substantially free of trehalose, sucrose, mannitol, sorbitol, methionine, histidine, glycine, and / or arginine.

[0039] In some embodiments, the aqueous formulation is substantially free of phosphates; for example, the aqueous formulation is substantially free of sodium phosphate and / or potassium phosphate. As used herein, in a composition (e.g., an aqueous formulation) “substantially free of” one or more specific compounds (e.g., histidine or sucrose, etc.), this compound may not be present in excess of trace amounts. In some embodiments, in a composition (e.g., an aqueous formulation) “substantially free of” one or more specific compounds, this compound may be present at a concentration of less than 5 mM, e.g., ≤1 mM, ≤0.5 mM, ≤0.1 mM, ≤0.05 mM or ≤0.01 mM. By way of illustration, in an aqueous formulation according to the present disclosure that is substantially free of histidine buffer salts, the histidine buffer salts may have a concentration of less than 5 mM, e.g., ≤1 mM, ≤0.5 mM, ≤0.1 mM, ≤0.05 mM or ≤0.01 mM in the aqueous formulation. In some embodiments, the aqueous formulation does not consist essentially of (i) 10 mM of L-histidine / L-histidine hydrochloride (L-His / L-His HCl), and (ii) 0.02% (w / v) of polysorbate 80.

[0040] In some embodiments, the aqueous formulation does not consist essentially of (i) 10 mM of L-histidine / L-histidine hydrochloride, and (ii) 0.02% (w / v) of polysorbate 80, and (iii) Fe at any one concentration of 0.1, 1 or 10 ppm 2+ and does not consist essentially of. In some embodiments, the aqueous formulation does not consist essentially of (i) 10 mM of sodium phosphate monobasic monohydrate / sodium phosphate dibasic anhydride, and (ii) 0.02% (w / v) of polysorbate 80. In some embodiments, the aqueous formulation does not consist essentially of (i) 25 mM of L-His / L-His HCl, (ii) 600 mM of sucrose, and (iii) 0.05% (w / v) of polysorbate 80. In some embodiments, the aqueous formulation does not consist essentially of (i) 10.5 mM of L-His / L-His HCl, (ii) 250 mM of sucrose, (iii) 0.02% (w / v) of polysorbate 80, and (iv) 1.4 mg / mL of rituximab. Rituximab (DrugBank Acc. No. DB00073) is a monoclonal anti-CD20 antibody.

[0041] In some embodiments, the aqueous formulation is essentially no different from (i) 10.5 mM L-His / L-His HCl, (ii) 250 mM sucrose, and (iii) 0.02% (w / v) polysorbate 80. It is understood that citrate is added to aqueous formulations to prevent the degradation of polysorbate (e.g., PS80) in the aqueous formulation. Therefore, the final concentration of citrate in the aqueous formulation may depend on the concentration of polysorbate (e.g., PS80). It is understood that the concentrations of citrate and polysorbate (e.g., PS80) in the aqueous formulation are selected according to the molar ratio of citrate to polysorbate (e.g., PS80) described herein. Therefore, it is understood that citrate is added to the aqueous formulation to prevent the degradation of polysorbate 20 in the aqueous formulation. Thus, the final concentration of citrate in the aqueous formulation may depend on the concentration of polysorbate 20. It is understood that the concentrations of citrate and polysorbate 20 in the aqueous formulation are selected according to the molar ratio of citrate to polysorbate 20 described herein. In some embodiments, the concentration of citrate in the aqueous formulation is less than 10 mM, preferably less than 5 mM, and more preferably less than 3 mM. If the concentration of citrate (e.g., concentration ≥ 5 mM) is high, protein particles may be formed and / or gelation may be promoted, for example, when a protein or peptide is added. In some embodiments, the concentration of citrate in the aqueous formulation is about 50 μM to about 2.5 mM. In some embodiments, the concentration of citrate in the aqueous formulation is about 250 μM to about 1 mM. In some embodiments, the concentration of citrate in the aqueous formulation is about 0.5 mM to about 1 mM.

[0042] In some embodiments, the citrate concentration is less than 2.5 mM. In contrast, for example, the concentration of citrate acting as a buffer in protein formulations is usually 10 mM to 20 mM, but at concentrations less than 2.5 mM, for example, citrate is usually unable to act as a buffer in aqueous formulations. Therefore, citrate preferably does not act as a buffer in aqueous formulations. Preferably, citrate acts as a stabilizer in aqueous formulations. In some embodiments, the concentration of citrate in the aqueous formulation is approximately 0.25 mM. In some embodiments, the concentration of citrate in the aqueous formulation is approximately 0.50 mM. In some embodiments, the concentration of citrate in the aqueous formulation is approximately 0.75 mM. In some embodiments, the concentration of citrate in the aqueous formulation is approximately 1 mM.

[0043] In some embodiments, the concentration of citrate in the aqueous formulation is about 250 μM to about 1 mM. In some embodiments, the concentration of citrate in the aqueous formulation is about 250 μM to about 750 μM. In some embodiments, the concentration of citrate in the aqueous formulation is about 250 μM to about 0.5 mM. In some preferred embodiments, the concentration of citrate in the aqueous formulation is about 0.5 mM. The aqueous formulations according to this disclosure may further comprise the substance of interest, for example, a substance useful for therapy, prevention, and / or diagnosis. Such substances include biomolecules, for example, proteins (e.g., peptides / polypeptides, complexes thereof), glycoproteins, lipoproteins, nucleic acids (e.g., polynucleotides / oligonucleotides), sugars, lipids (fatty acids, glycerides), phospholipids, glycolipids, etc., and complexes thereof. In some embodiments, the aqueous formulations according to this disclosure comprise peptides / polypeptides or complexes thereof (e.g., those listed herein) for use in therapy, prevention, and / or diagnosis.

[0044] In some embodiments, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 1 × 10⁻⁶ -5 mg / mL to approximately 15 mg / mL, for example, approximately 1 × 10 -5mg / mL ~ approx. 1×10 -4 mg / mL, approximately 1×10 -4 The concentration is one of the following ranges: mg / mL to approximately 0.01 mg / mL, approximately 0.01 mg / mL to approximately 0.1 mg / mL, approximately 0.1 mg / mL to approximately 1 mg / mL, approximately 1 mg / mL to approximately 5 mg / mL, approximately 5 mg / mL to approximately 10 mg / mL, and approximately 10 mg / mL to approximately 15 mg / mL. In some embodiments, the concentration of the substance is approximately 1 × 10⁻⁶ -5 mg / mL, approximately 1×10 -4The concentrations are mg / mL, approximately 0.01 mg / mL, approximately 0.1 mg / mL, approximately 1 mg / mL, approximately 5 mg / mL, approximately 10 mg / mL, or approximately 15 mg / mL. In some embodiments, the concentration of the substance in the aqueous formulation / composition according to this disclosure is at least 0.1 mg / mL, at least 1 mg / mL, at least 5 mg / mL, or at least 10 mg / mL. In some embodiments, the concentration of the substance in the aqueous formulation / composition according to this disclosure is approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, or approximately 30 mg / mL. In some embodiments, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 10 mg / mL to about 100 mg / mL, for example, one of the following concentrations: about 10 mg / mL to about 20 mg / mL, about 20 mg / mL to about 30 mg / mL, about 30 mg / mL to about 40 mg / mL, about 40 mg / mL to about 50 mg / mL, about 50 mg / mL to about 60 mg / mL, about 60 mg / mL to about 70 mg / mL, about 70 mg / mL to about 80 mg / mL, about 80 mg / mL to about 90 mg / mL, and about 90 mg / mL to about 100 mg / mL. Preferably, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 10 mg / mL to about 75 mg / mL. More preferably, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 10 mg / mL to about 50 mg / mL. Even more preferably, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 10 mg / mL to about 30 mg / mL. Even more preferably, the concentration of the substance (e.g., peptide / polypeptide) in the aqueous formulation / composition according to this disclosure is about 10 mg / mL to about 25 mg / mL. In some embodiments, the concentration of the substance in the aqueous formulation / composition according to this disclosure is about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, or about 70 mg / mL. In some embodiments, the concentration of the substance is 50 mg / mL. In some embodiments, the concentration of the substance in the aqueous formulation / composition according to this disclosure is about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL.In some embodiments, the concentration of the substance is 100 mg / mL.

[0045] In some embodiments, the aqueous formulation is - Approximately 0.2mg·ml -1 Polysorbates (e.g., PS80 or PS20, etc.), - Approximately 0.9% (w / v) sodium chloride, - water, - 0.25 mM to 1 mM, preferably about 0.5 mM of citrate, and - Optionally, a pharmaceutically acceptable carrier, excipient, or adjuvant. - Optionally, peptides / polypeptides for therapeutic, prophylactic, and / or diagnostic use, which may be at concentrations defined above herein. It becomes essentially. Preferably, the aqueous formulation is - Approximately 0.2mg·ml -1 Polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - Water, and - 0.25 mM to 1 mM, preferably about 0.5 mM citrate It becomes essentially.

[0046] Prevention of polysorbate degradation This disclosure provides an aqueous formulation that prevents the degradation of polysorbate (e.g., polysorbate 80 or polysorbate 20). That is, the polysorbate is stable in the aqueous formulation so that the level of intact polysorbate does not decrease significantly in the aqueous formulation (e.g., during storage and / or exposure). In some embodiments, the present disclosure provides an aqueous formulation that prevents the degradation of PS20. That is, PS20 is stable in the aqueous formulation so that the level of intact PS20 does not decrease significantly in the aqueous formulation (for example, during storage). In some embodiments, the decomposition of polysorbate includes the oxidation of polysorbate (e.g., polysorbate 80). In some embodiments, the decomposition of polysorbate 20 includes the oxidation of polysorbate 20.

[0047] The oxidation pathway targets the hydrocarbon chain double bonds of unsaturated fatty acids and / or the polyoxyethylene (POE) chain of polysorbate 20, as summarized by Donbrow et al. (26). The oxidation of polysorbate 80 involves two oxidation pathways. The head group of nonionic surfactants is oxidized by free radical species, resulting in fragmentation of the head group. Free radical species can also oxidize any unsaturated fatty acids bound to the head group, producing various decomposition and fragmentation species (Gopalrathnam et al. (2018)) (47). Oxidative degradation can occur as a result of the presence of reactive oxygen species (ROS) in aqueous formulations. ROS can be generated in the presence of hydrogen peroxide (H2O2). In some embodiments, the decomposition of polysorbates (e.g., PS80, PS20, etc.) includes decomposition as a result of exposure (e.g., photo-oxidation).

[0048] In some embodiments, the degradation of polysorbate (e.g., PS80) includes degradation in the presence of peptides / polypeptides or complexes thereof as described herein, for example, in compositions as described herein. In some embodiments, the degradation of polysorbate 20 includes, for example, the degradation of polysorbate 20 in a composition described herein, in the presence of peptides / polypeptides or complexes thereof described herein. In some embodiments, the decomposition of polysorbate (e.g., polysorbate 80) includes the decomposition of polysorbate (e.g., polysorbate 80) in the presence of metal ions. In some embodiments, the decomposition of polysorbate 20 includes the decomposition of polysorbate 20 in the presence of metal ions. Therefore, in some embodiments, the decomposition of PS20 includes decomposition mediated by the generation of metal ion-dependent hydroxyl radicals. Metal ions, such as copper ions and iron ions, are known to be involved in Fenton-type ROS generation. In certain embodiments, the metal ion is iron ion, for example, Fe 2+ That is the case.

[0049] Metal ions can originate from any substance / molecule containing metal ions. In some embodiments, the source of metal ions may be a vessel / container or support for an aqueous formulation. For example, the source of metal ions may be a metal ion-containing vessel / container for preparing / containing / storing / serving the aqueous formulation, such as a stainless steel vessel / container. In some embodiments, the source of metal ions may be a carrier, excipient, or adjuvant used with or contained in the aqueous formulation. In some embodiments, preventing the degradation of polysorbate (e.g., PS80) includes preventing the degradation of polysorbate (e.g., PS80) in an aqueous formulation or composition in a container / vessel containing or made from stainless steel. In some embodiments, preventing the degradation of PS20 includes preventing the degradation of PS20 in an aqueous formulation or composition in a container / vessel containing or made from stainless steel. In some embodiments, the aqueous formulation or composition is contained in a container (e.g., a syringe) containing a stainless steel needle.

[0050] In some embodiments, the aqueous formulation or composition is contained in a glass container, such as a glass vial, such as a type I borosilicate glass vial. In some embodiments, the aqueous formulation is provided in a container / container containing or made from plastic. Plastics generally include organic polymers and include acrylic, polyester, silicone, polyurethane, and halogenated plastics. In some embodiments, the plastics according to this disclosure are or contain vinyl polymers or vinyl copolymers, polyethylene (PE), polypropylene (PP), polystyrene, polyvinyl chloride (PVC), polyvinyl esters, polyvinyl acetate (PVAc), polyacrylonitrile, polyolefin (PP+PE), and ethyl-vinyl acetate (EVA; copolymer of ethylene and vinyl acetate). In some embodiments, the aqueous formulation is provided in a container / container containing or made from EVA, for example, the container may be provided in an injectable bag, such as a Flexboy® bag.

[0051] Preventing the degradation of polysorbates (e.g., PS80) may involve maintaining the level of (intact) polysorbate in aqueous formulations over a specific period. An example LCMS-based method for quantifying PS80 levels is described in Gopalrathnam et al. (2018)(47), which is incorporated herein by reference in its entirety. An example method for quantifying polysorbate levels is a fluorescence micelle assay (FMA). Such an assay is described in Lippold et al. (2017)(41), which is incorporated herein by reference in its entirety. Preventing the degradation of PS20 may involve maintaining (intact) PS20 levels in aqueous formulations over a specific period. An example of an LCMS-based method for quantifying PS20 levels is a fluorescence micelle assay (FMA). Such an assay is described by Lipppold et al. (2017)(41), which is incorporated herein by reference in its entirety.

[0052] In some embodiments, the period is one of the following: approximately 2 weeks to approximately 36 months, for example, approximately 2 weeks to approximately 1 month, approximately 1 month to approximately 2 months, approximately 2 months to approximately 3 months, approximately 3 months to approximately 4 months, approximately 4 months to approximately 5 months, approximately 5 months to approximately 6 months, approximately 6 months to approximately 7 months, approximately 7 months to approximately 8 months, approximately 8 months to approximately 9 months, approximately 9 months to approximately 10 months, approximately 11 months to approximately 12 months, approximately 12 months to approximately 15 months, approximately 15 months to approximately 18 months, approximately 18 months to approximately 21 months, approximately 21 months to approximately 24 months, approximately 24 months to approximately 27 months, approximately 27 months to approximately 30 months, approximately 30 months to approximately 33 months, or approximately 33 months to approximately 36 months. In some cases, the duration is at least about two weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about fifteen months, at least about eighteen months, at least about twenty-one months, at least about twenty-four months, at least about twenty-seven months, at least about thirty months, at least about thirty-three months, or at least about thirty-six months.

[0053] In some embodiments, preventing the decomposition of PS20 includes maintaining the level of PS20 in the aqueous formulation for a given period of time (e.g., the period described herein) at a given temperature. In some embodiments, preventing the decomposition of polysorbate (e.g., PS80) includes maintaining the essential level of polysorbate (e.g., PS80) in the aqueous formulation for a given period of time (e.g., the period described herein) at a given temperature. In some embodiments, the given temperature is one of the following temperatures: about 2°C to about 60°C, for example, about 2°C to about 5°C, about 5°C to about 10°C, about 10°C to about 15°C, about 15°C to about 20°C, about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, about 35°C to about 40°C, about 40°C to about 45°C, about 45°C to about 50°C, about 50°C to about 55°C, or about 55°C to about 60°C. In some embodiments, the temperature is approximately 2°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0054] In some embodiments, maintaining the level of PS20 includes maintaining at least 95%, at least 90%, or at least 85% of the initial amount of PS20 in the aqueous formulation or composition. In some embodiments, maintaining the level of polysorbate (e.g., PS80) includes maintaining at least 95%, at least 90%, or at least 85% of the initial amount of polysorbate (e.g., PS80) in the aqueous formulation or composition. In some embodiments, the aqueous formulation or composition contains hydrogen peroxide (to prevent the decomposition of polysorbate, particularly PS20, in the presence of hydrogen peroxide). In some embodiments, the concentration of hydrogen peroxide is about 1 ppm to about 1000 ppm, for example, one of the following concentrations: 1 ppm to 100 ppm, 100 ppm to 250 ppm, 250 ppm to 500 ppm, 500 ppm to 750 ppm, and 750 ppm to 1000 ppm. In some embodiments, the concentration of hydrogen peroxide is 1, 100, 250, 500, 750, or 1000 ppm. However, H2O2 is usually not required. In the appended examples, H2O2 was added to enhance stress during stability testing. Preferably, the aqueous formulation or composition does not contain hydrogen peroxide.

[0055] In some embodiments, the aqueous formulation or composition contains polysorbate, particularly at least 95%, at least 90%, or at least 85% of the initial amount of PS20, after incubation at 40°C or below, for example, for a period of about 0.5 to about 12 months at about 40°C. In some embodiments, the aqueous formulation or composition contains polysorbate, particularly at least 95%, at least 90%, or at least 85% of the initial amount of PS20, after incubation at about 25°C for a period of about 0.5 to about 12 months. In some embodiments, the aqueous formulation or composition contains polysorbate, particularly at least 95%, at least 90%, or at least 85% of the initial amount of PS20, after incubation for one of the following periods at 40°C or below, e.g., about 40°C or about 25°C: about 2 weeks to about 1 month, about 1 month to about 2 months, about 2 months to about 3 months, about 3 months to about 4 months, about 4 months to about 5 months, about 5 months to about 6 months, about 6 months to about 7 months, about 7 months to about 8 months, about 8 months to about 9 months, about 9 months to about 10 months, and about 11 months to about 12 months. In some embodiments, the period is at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.

[0056] In some embodiments, the aqueous formulation or composition contains, for example, at least 95% of the initial amount of polysorbate, particularly PS20, after a period of about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 12 months, under conditions specified above herein (e.g., temperature and / or presence of hydrogen peroxide). In some embodiments, the aqueous formulation or composition contains, for example, at least 90% of the initial amount of polysorbate, particularly PS20, after a period of about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 12 months, under conditions specified above herein (e.g., temperature and / or presence of hydrogen peroxide). In some embodiments, the aqueous formulation or composition contains, for example, at least 85% of the initial amount of polysorbate, particularly PS20, after a period of about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 12 months, under conditions specified above herein (e.g., temperature and / or presence of hydrogen peroxide).

[0057] In some embodiments, the aqueous formulation or composition is provided in the container described herein. In some embodiments, the aqueous formulation or composition is stored at a relative humidity of about 75%. In some embodiments, preventing the degradation of PS20 involves reducing the level of PS20 oxidation products in the aqueous formulations of this disclosure compared to the level of citrate-free aqueous formulations. In some embodiments, the oxidation products include one or more of the following: monoester of lauric acid having five POE subunits (POE5 Mono), diester of formic acid and lauric acid having five POE subunits (POE5 Di), monoester of lauric acid having six POE subunits (POE6 Mono), and diester of formic acid and lauric acid having six POE subunits (POE6 Di). The structures of such oxidation markers are shown in Table 3. Suitable methods for quantifying the oxidation products include ultra-high performance liquid chromatography (UPLC-MS) combined with mass spectrometry.

[0058] The oxidation products of PS80 have been reported in Hvattum et al. (2012)(49) and Borisov et al. (2015)(50), and these disclosures are incorporated herein by reference in their entirety. In some embodiments, prevention of PS80 degradation involves reducing the level of PS80 oxidation products in the aqueous formulations of this disclosure compared to the level of citrate-free aqueous formulations. In some embodiments, the oxidation products include 9-oxo-C9:0-esters and / or hydroxy-C18:1-esters. Suitable methods for quantifying the oxidation products include ultra-high performance liquid chromatography (UPLC-MS) combined with mass spectrometry. Therefore, compared to aqueous formulations or compositions that do not contain citrate, aqueous formulations or compositions obtained by the method of this disclosure, maintained under the same conditions (e.g., the same temperature, the same relative humidity, the same concentration of oxidizing agents, etc.), may exhibit one or both of the following characteristics: Including a higher level of PS20 over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months, Optionally, include lower levels of PS20 oxidation markers over a specific period, e.g., 1, 2, 3, 4, 6, 12, 18, 24, or 36 months.

[0059] Furthermore, compared to aqueous formulations or compositions that do not contain citrate, aqueous formulations or compositions obtained by the method of this disclosure, maintained under the same conditions (e.g., the same temperature, the same relative humidity, the same concentration of oxidizing agents, under exposure, etc.), may exhibit one or more of the following characteristics: A higher level of polysorbate is included over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months. Optionally, include lower levels of polysorbate oxidation markers over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months. A higher level of PS80 is included over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months. Optionally, include lower levels of PS80 oxidation markers over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months. Optionally, over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months, if the turbidity level is the same, Optionally, over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months, include microparticles of significantly the same concentration. Optionally, over a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months, including lower concentrations of microparticles, Optionally, maintain the level of photostability of the polysorbate and / or polypeptide in the aqueous formulation for a specific period, for example, 1, 2, 3, 4, 6, 12, 18, 24, or 36 months.

[0060] composition As described herein, aqueous formulations may be used as diluents for compositions. The diluent is a solution used to prepare a composition or a peptide / polypeptide or complex thereof, such as an antibody or an antigen-binding fragment, or a formulation. For example, a diluent may be used to prepare a composition for administration. The compositions of this disclosure may comprise peptides / polypeptides or complexes thereof as described herein, particularly antibodies (i.e., immunoglobulins) or antigen-binding fragments thereof. In some embodiments, the compositions are provided in lyophilized form. In this case, an aqueous formulation may be used to resuspend the lyophilized composition. Alternatively, the lyophilized composition may first be resuspended in a separate medium and then diluted with an aqueous formulation as described herein to provide, for example, a peptide / polypeptide or complex thereof (e.g., an antibody or antigen-binding fragment) at a concentration desired for administration. In some embodiments, the compositions comprise the substance of interest (e.g., peptides / polypeptides) at a concentration specified herein.

[0061] In some embodiments, the present disclosure provides a composition comprising a polysorbate (e.g., PS80, PS20, etc.), a citrate, and sodium chloride, wherein the molar ratio of citrate to polysorbate in the composition is about 1.5 to about 7, and the composition is provided in a lyophilized form. In some embodiments, the molar ratio is about 1.6 to about 7. In some embodiments, the molar ratio is about 3 to about 6. In preferred embodiments, the molar ratio of citrate to polysorbate in the composition is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the composition does not contain histidine. In some embodiments, the composition further comprises a metal ion. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. In some embodiments, the composition is provided in a container comprising a glass container, a stainless steel container, or a plastic container. The composition may further comprise peptides / polypeptides for use in therapy, prevention, and / or diagnosis. In some embodiments, the peptides / polypeptides for use in therapy, prevention, and / or diagnosis are selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0062] This disclosure further provides a method for preparing a composition, comprising the step of contacting a peptide / polypeptide or complex thereof for use in therapy, prevention, and / or diagnosis with an aqueous formulation. The peptide / polypeptide or complex thereof described herein may be formulated as a pharmaceutical composition or pharmaceutical for clinical use and may contain pharmaceutically acceptable carriers, excipients, or adjuvants. In some embodiments, the aqueous formulation or composition comprises a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0063] The compositions / aqueous formulations of this disclosure include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, methylparaben, propylparaben), antioxidants (e.g., vitamin C, selenium, cysteine, methionine, methylparaben, propylparaben), and antioxidants (e.g., vitamin C). The aqueous formulation / composition may contain trehalose, vitamin E, vitamin C, retinyl palmitate, selenium, lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or coloring agents (e.g., titanium dioxide). In some embodiments, the aqueous formulation / composition does not contain sucrose. In some embodiments, the aqueous formulation / composition does not contain trehalose, sucrose, mannitol, sorbitol, methionine, histidine, glycine, and / or arginine.

[0064] When used herein, the term “pharmaceutically acceptable” refers to compounds, components, materials, compositions, dosage forms, etc. These are within the bounds of sound medical judgment, free from excessive toxicity, irritation, allergic reactions, and other problems and complications, and are suitable for use in contact with the tissues of the subject (e.g., human subjects) in a reasonable risk-benefit ratio. Furthermore, each carrier, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking substance, colorant, flavoring substance, or sweetener of a composition according to this disclosure must be “acceptable” in the sense that it is compatible with the other components of the formulation. Suitable carriers, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking substances, colorants, flavoring substances, or sweeteners are listed in standard pharmaceutical texts, e.g., Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23 rd It can be found in Edition (2020), Academic Press.

[0065] The compositions of this disclosure (e.g., pharmaceutical compositions) may be formulated or prepared according to the methods of this disclosure for intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. In some embodiments, the compositions may be formulated / prepared for administration by injection or infusion or by oral ingestion. Accordingly, the aqueous formulations of this disclosure may be suitable for intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. In some embodiments, the aqueous formulations of this disclosure may be suitable for administration by injection or infusion. In some embodiments, the composition (e.g., a pharmaceutical composition) is formulated / prepared for injection or infusion into, for example, blood vessels, tissues / organs of interest, or tumors.

[0066] In some embodiments, the composition is prepared by diluting it with the aqueous formulations described herein. Depending on the subsequent use of the composition, an appropriate dilution may be used. For example, in some embodiments, the composition may be diluted 10-fold, 100-fold, 10-fold, etc. 3 double, 10 4 double, 10 5 double or 10 6 Dilute by a factor of two. For example, the preparation of a composition for administration by injection may require a dilution ratio of, for example, 100 times. Furthermore, this disclosure provides a method for producing pharmaceutically useful compositions, and such a production method is A step of generating peptides / polypeptides or complexes thereof for use in therapies, preventive and / or diagnostics as described herein, and / or A step of mixing a peptide / polypeptide or a complex thereof with the aqueous formulation described herein. It may include one or more steps selected from the following.

[0067] material The aqueous formulations of this disclosure are useful as diluents for the substance of interest, for example, the substance useful in the above-described therapeutic, prophylactic, and / or diagnostic applications. In some embodiments, the substance is a peptide / polypeptide or complex thereof suitable for use in therapy, prevention, and / or diagnosis. The peptide / polypeptide or complex thereof may be contained in the compositions described herein and / or may be contacted with the aqueous formulations of this disclosure using the methods described herein. Peptides / polypeptides or complexes thereof suitable for use in therapy, prevention, and / or diagnosis are any peptide / polypeptide (e.g., proteins) or complexes thereof that possess therapeutic, prophylactic, or diagnostic properties. Such peptides / polypeptides and peptide / polypeptide complexes are known to those skilled in the art, and for example, Dimitrov et al. Methods Mol Biol. 2012, 899: 1-26 (incorporated herein by reference in its entirety) present a review of proteins suitable for use in therapy / prevention.

[0068] Peptide / polypeptide complexes are characterized by protein-protein interactions between their constituent polypeptides / peptides. In some embodiments, the association includes non-covalent interactions (electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces) and / or covalent interactions (e.g., disulfide bonds). Exemplarily, a peptide / polypeptide complex considered in accordance with this disclosure comprises an IgG antibody, which includes four polypeptide chains that associate via protein-protein interactions to form a polypeptide complex. In some embodiments, the peptide / polypeptide or complex thereof is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. Antigen-binding peptides / polypeptides and antigen-binding peptide / polypeptide complexes refer to peptides / polypeptides and peptide / polypeptide complexes that bind to a given target antigen. Such peptides / polypeptides and peptide / polypeptide complexes include antibodies (i.e., immunoglobulins including monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies, etc.)), antibody fragments (antigen-binding fragments, e.g., Fv fragments, Fab fragments, F(ab')2 fragments, and F(ab') fragments), and antibody-derived molecules (including scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH), etc.).

[0069] In some embodiments, the antigen-binding polypeptide is a monoclonal antibody. In some embodiments, the antigen-binding peptide / polypeptide includes or consists of peptide aptamers, thioredoxins, monobodies, anticalin, Knitz domains, avimers, Nottin, fynomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), Obodies, or fibronectin, for example, those described in the review Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101, which is incorporated herein by reference in whole (also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011)). (See 3:38-48).

[0070] In some embodiments, the antigen-binding polypeptide comprises or consists of the antigen-binding region of an antibody (e.g., the antigen-binding fragment of an antibody). The antigen-binding polypeptide of this disclosure preferably comprises the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) of an antibody that bind to a target antigen. The antigen-binding domain formed by the VH and VL may also be referred to herein as the Fv region. In some embodiments, the antigen-binding polypeptide is or comprises the Fv of the antibody (e.g., provided as scFv). In some embodiments, the antigen-binding polypeptide is or comprises the Fab region of the antibody. In some embodiments, the antigen-binding polypeptide is or comprises the entire antibody (i.e., including the variable region and the constant region). An antigen-binding polypeptide may be or may comprise an antigen-binding polypeptide complex. An antigen-binding polypeptide may comprise two or more polypeptides that together form an antigen-binding moiety. Polypeptides may associate covalently or non-covalently. In some embodiments, polypeptides form a part of a larger polypeptide comprising this polypeptide (for example, in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).

[0071] An antigen-binding polypeptide may refer to an IgG-like antigen-binding polypeptide comprising two or more polypeptides (e.g., 2, 3, 4, 6, or 8 polypeptides) via non-covalent or covalent bonds, such as two heavy-chain polypeptides and two light-chain polypeptides. The antigen-binding polypeptides of this disclosure may be designed and prepared using sequences of monoclonal antibodies (mAbs) capable of binding to a given target antigen. Antigen-binding regions of antibodies, such as single-strand variable fragments (scFv), Fab fragments, and F(ab')2 fragments, may also be used / provided. An "antigen-binding region" is any fragment of an antibody that binds to a specific target. Antibodies typically contain six complementarity-determining regions (CDRs): three heavy chain variable (VH) regions: HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain variable (VL) regions: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the antibody paratope, which is a portion of the antibody that binds to the target antigen. The VH and VL regions contain framework regions (FRs) on both sides of each CDR, which form the scaffold of the CDR. The VH region contains the following structure from the N-terminus to the C-terminus: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term, and the VL region contains the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C term.

[0072] The VL region and light chain constant (CL) region, as well as the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of the antibody, together constitute the Fab region. In some embodiments, the antigen-binding polypeptide includes a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH (e.g., a VL-CH1 fusion polypeptide). That is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH region, CH1 region, VL region, and CL region of the Fab or CrossFab are linked by a linker region to provide a single polypeptide, i.e., a single-stranded Fab (scFab) or single-stranded CrossFab (scCrossFab).

[0073] In some embodiments, the antigen-binding polypeptides described herein include or consist of a whole antibody. As used herein, “whole antibody” refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Various types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41–S52, which is incorporated herein by reference in its entirety. G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa containing two heavy chains and two light chains. The heavy chains, from the N-terminus to the C-terminus, contain a heavy chain constant region with three constant domains (CH1, CH2, and CH3) after VH, and similarly the light chains contain CL after VL. Immunoglobulins can be classified into IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM depending on the heavy chain. The light chains can be kappa (κ) or lambda (λ).

[0074] In some embodiments, the antigen-binding polypeptide includes or comprises IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In some embodiments, the antigen-binding polypeptide of the present disclosure includes an Fc region. As used herein, “Fc region” refers to a polypeptide complex formed by the interaction of two polypeptides, in which each polypeptide contains the CH2-CH3 region of the constant sequence of an immunoglobulin (Ig) heavy chain.

[0075] In this specification, "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH2 domain is the Ig region formed by positions 231 to 340 of the immunoglobulin constant domain. "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH3 domain is the Ig region formed by positions 341 to 447 of the immunoglobulin constant domain. "CH2-CH3 region" refers to the amino acid sequences corresponding to the CH2 and CH3 domains of immunoglobulin (Ig). According to the EU numbering scheme described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH2-CH3 region is an Ig region formed from positions 231 to 447 of the constant-state immunoglobulin domain.

[0076] In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region according to this disclosure corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0077] The Fc region enables interaction with Fc receptors and other molecules of the immune system, thereby producing functional effects. Fc-mediated effector functions are described, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (the entire article is incorporated herein by reference), and are brought about by Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interaction between the Fc region and Fc receptors expressed by immune cells, recruitment of complement pathway components by binding of the Fc region to complement protein C1q, and consequently activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane invasion complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and antigen presentation. In some embodiments of this disclosure, the peptides / polypeptides suitable for use in therapy, prevention, and / or diagnosis are Fc fusion proteins. Fc fusion proteins consist of an immunoglobulin Fc region that directly binds to another peptide. Various types of Fc fusion proteins and their structures are described, for example, in Czajkowsky et al. EMBO Mol Med (2012) 4, 1015-1028, which is incorporated herein by reference in its entirety.

[0078] Method and Use Furthermore, this disclosure provides a method for preventing the degradation of polysorbate 20 in an aqueous formulation, comprising the step of adding citrate until the final molar ratio of citrate to polysorbate 20 is about 1.5 to about 7. Suitable molar ratios of citrate to polysorbate 20, the concentration of PS20, and the concentration of citrate are disclosed above in this specification. The step of "adding citrate" may include the step of adding citric acid or a salt thereof. For example, to prepare an aqueous formulation or composition according to the present disclosure, for example, citric acid monohydrate, sodium citrate dihydrate, or trisodium citrate may be added. Furthermore, the use of citrate is provided to prevent the degradation of polysorbate 20 in aqueous formulations, where the final molar ratio of citrate to polysorbate 20 is approximately 1.5 to approximately 7. Suitable molar ratios of citrate to polysorbate 20, the concentration of PS20, and the concentration of citrate are disclosed above herein.

[0079] In some embodiments, the use or method is (a) a step of preparing an aqueous formulation according to the present disclosure, and (b) The step of contacting a substance described herein (e.g., a peptide / polypeptide described herein) with an aqueous formulation. It also includes. In some embodiments, step (b) includes a step of diluting the substance with an aqueous formulation. In some embodiments, step (b) includes a step of adjusting the concentration of the substance in the aqueous formulation. This disclosure further provides a method for preparing an aqueous formulation, comprising the step of contacting an aqueous solution containing polysorbate 20 with citrate until the final molar ratio of citrate to polysorbate 20 is about 1.5 to about 7. Suitable final molar ratios of citrate to polysorbate 20, the final concentration of PS20, and the final concentration of citrate are disclosed herein.

[0080] This disclosure further provides a method for preparing an aqueous formulation, comprising the step of contacting an aqueous solution containing polysorbate (e.g., PS80) with citrate until the final molar ratio of citrate to polysorbate is about 1.5 to about 7, preferably about 1.6 to about 7. A suitable final molar ratio of citrate to polysorbate, e.g., citrate to PS80 or polysorbate 20, the final concentration of PS, e.g., the final concentration of PS80 or PS20, and the final concentration of citrate are disclosed herein. The uses or methods relating to this disclosure may further include steps for incubating, storing, or maintaining an aqueous formulation. The aqueous formulation may be incubated / stored / maintained for a specified period and / or under certain conditions (e.g., at a specific temperature) as described herein. Furthermore, the use of the aqueous formulations of this disclosure as a diluent for a substance or composition, for example, as a diluent for the substance / composition described herein, is also provided. That is, in some embodiments, the aqueous formulation is provided as a diluent. Use may include the step of diluting the substance / composition for administration. The Disclosure further provides a method for preparing a composition, for example, a method for preparing a composition for administration, which includes the step of contacting a substance described herein with an aqueous formulation of the Disclosure.

[0081] kit Furthermore, this disclosure provides kits of parts. The kits provided herein may include, in whole or in part, the components for carrying out the methods described herein. The kit may have at least one container containing a predetermined amount of the aqueous formulation or composition described herein. Suitable containers are those described herein. In some embodiments of this disclosure, a kit of parts is provided. In some embodiments, the kit may include aqueous formulations and / or compositions described herein, which may be provided in predetermined quantities. Furthermore, the present invention provides a kit comprising a pharmaceutical composition and a diluent for diluting the pharmaceutical composition, wherein the diluent is an aqueous formulation of the present disclosure.

[0082] The kit may provide the pharmaceutical compositions described herein, along with instructions for administering to a patient for the treatment of a specific disease / condition (e.g., the disease / condition described herein, e.g., cancer). The kit may further include reagents, buffers, and / or reference materials required to carry out the methods according to this disclosure, for example, a method for preparing a pharmaceutical composition for administration. The kit according to this disclosure may include instructions for use, for example, in the form of a booklet or leaflet. The instructions may include protocols for carrying out any one or more of the methods described herein.

[0083] Description with item number The following numbered sections describe specific aspects and embodiments of the present invention. 1. An aqueous formulation containing polysorbate 20, citrate, and sodium chloride. 2. The aqueous formulation described in item 1, wherein the concentration of citrate in the aqueous formulation is approximately 50 μM to approximately 2.5 mM. 3. The aqueous formulation according to item 1 or item 2, wherein the concentration of citrate in the aqueous formulation is approximately 250 μM to approximately 1 mM. 4. An aqueous formulation according to any one of items 1 to 3, wherein the concentration of polysorbate 20 in the aqueous formulation is approximately 0.02% (w / v). 5. An aqueous formulation according to any one of items 1 to 4, wherein the concentration of sodium chloride in the aqueous formulation is approximately 0.9% (w / v). 6. An aqueous formulation according to any one of items 1 to 5, wherein the molar ratio of citrate to polysorbate 20 in the aqueous formulation is approximately 1.5 to approximately 7. 7. An aqueous formulation according to any one of items 1 to 6, wherein the molar ratio of citrate to polysorbate 20 is approximately 3 to approximately 6. 8. An aqueous formulation according to any one of items 1 to 7, wherein the molar ratio of citrate to polysorbate 20 is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. 9. An aqueous formulation according to any one of items 1 to 8, which does not contain histidine. 10. An aqueous formulation according to any one of items 1 to 9, further comprising metal ions. 11. An aqueous formulation according to any one of items 1 to 10, wherein the concentration of sodium chloride in the aqueous formulation is approximately 0.9% (w / v), and the molar ratio of citrate to polysorbate 20 in the aqueous formulation is approximately 3 to approximately 6. 12. An aqueous preparation according to any one of items 1 to 11, comprising essentially polysorbate 20, citrate, water, and sodium chloride. 13. Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - Water, and - 0.25 mM to 1 mM citrate An aqueous formulation, essentially comprising one of items 1 to 12. 14. Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - Water, and - Approximately 0.5 mM citrate The aqueous formulation described in item 13, which is essentially derived from the above. 15. An aqueous preparation described in any one of items 1 to 14, suitable for intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. 16. An aqueous formulation according to any one of items 1 to 15, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant. 17. An aqueous formulation according to any one of items 1 to 16, provided in a glass container, a stainless steel container or a container containing plastic. 18. An aqueous formulation according to any one of items 1 to 17, further comprising a peptide / polypeptide for use in therapy, prevention, and / or diagnosis. 19. An aqueous formulation according to item 18, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. 20. A method for preventing the decomposition of polysorbate 20 in an aqueous formulation, comprising the step of adding citrate until the final molar ratio of citrate to polysorbate 20 is about 1.5 to about 7. 21. The method according to item 20, wherein the aqueous preparation comprises sodium chloride. 22. The method according to item 20 or 21, wherein the aqueous formulation is an aqueous formulation as defined in any one of items 1 to 19. 23. The method according to any one of items 20 to 22, wherein the decomposition of polysorbate 20 includes oxidation of polysorbate 20. 24. The method according to any one of items 20 to 23, wherein the decomposition of polysorbate 20 includes the decomposition of polysorbate 20 in the presence of metal ions. 25. The method according to any one of claims 20 to 24, wherein the degradation of polysorbate 20 comprises the degradation of polysorbate 20 in the presence of peptides / polypeptides for use in therapy, prevention, and / or diagnosis. 26. The method according to item 25, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. 27. The method according to any one of items 20 to 26, wherein the aqueous formulation is provided in a container including a glass container, a stainless steel container or a plastic container. 28. The method according to any one of items 20 to 27, wherein the molar ratio of citrate to polysorbate 20 is approximately 3 to approximately 6. 29. The method according to any one of items 20 to 28, wherein the molar ratio of citrate to polysorbate 20 is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. 30. The method according to any one of claims 20 to 29, wherein the aqueous formulation comprises about 0.02% (w / v) of polysorbate 20. 31. Use of citrate to prevent the degradation of polysorbate 20 in aqueous formulations, where the final molar ratio of citrate to polysorbate 20 is approximately 1.5 to approximately 7. 32. The use of the aqueous formulation as described in item 31, wherein the aqueous formulation contains sodium chloride. 33. The use described in paragraph 32, wherein the aqueous formulation is an aqueous formulation as defined in any one of paragraphs 1 to 19. 34. Use according to any one of items 31 to 33, wherein the decomposition of polysorbate 20 includes oxidation of polysorbate 20. 35. Uses of any one of items 31 to 34, wherein the decomposition of polysorbate 20 includes the decomposition of polysorbate 20 in the presence of metal ions. 36. Uses of any one of the items 31 to 35, wherein the degradation of polysorbate 20 includes the degradation of polysorbate 20 in the presence of peptides / polypeptides for use in therapy, prophylaxis, and / or diagnostics. 37. Uses of peptides / polypeptides for therapeutic, prophylactic, and / or diagnostic purposes as described in item 36, selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. 38. The use described in any one of paragraphs 31 to 37, wherein the aqueous formulation is provided in a container containing glass, stainless steel, or plastic. 39. Use as described in any one of items 31 to 38, wherein the molar ratio of citrate to polysorbate 20 is approximately 3 to approximately 6. 40. Use as described in any one of items 31 to 39, wherein the molar ratio of citrate to polysorbate 20 is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. 41. Use according to any one of items 31 to 40, wherein the aqueous formulation contains approximately 0.02% (w / v) of polysorbate 20. 42. Use of an aqueous formulation as a diluent for a composition, as described in any one of items 1 to 17. 43. The use described in item 42, wherein the composition comprises peptides / polypeptides for use in therapy, prevention, and / or diagnosis. 44. Uses of peptides / polypeptides for therapeutic, prophylactic, and / or diagnostic use as described in item 43, selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. 45. A method for preparing a composition, comprising the step of contacting a peptide / polypeptide for use in therapy, prevention, and / or diagnosis with an aqueous formulation described in any one of items 1 to 17. 46. ​​The method according to item 45, wherein the composition is a pharmaceutical composition for administration. 47. The method according to item 46, wherein the administration is intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal. 48. Compositions obtained or obtainable by any one of the methods described in Sections 45 to 47. 49. A composition comprising polysorbate 20, citrate, and sodium chloride, A composition having a molar ratio of citrate to polysorbate 20 of about 1.5 to about 7, and provided in a lyophilized form. 50. The composition according to item 49, wherein the molar ratio of citrate to polysorbate 20 in the composition is about 3 to about 6. 51. The composition according to claim 49 or claim 50, wherein the molar ratio of citrate to polysorbate 20 in the composition is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. 52. A composition according to any one of items 49 to 51, which does not contain histidine. 53. A composition according to any one of items 49 to 52, further comprising a metal ion. 54. A composition according to any one of claims 49 to 53, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant. 55. The composition according to any one of items 49 to 54, provided in a container including a glass container, a stainless steel container, or a plastic container. 56. The composition according to any one of claims 49 to 55, further comprising peptides / polypeptides for use in therapy, prevention, and / or diagnosis. 57. The composition according to item 56, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. 58. A kit comprising an aqueous formulation as described in any one of sections 1 to 19 or a composition as described in section 48. 59. A kit comprising an aqueous formulation as described in any one of items 1 to 17 or a composition as described in any one of items 49 to 55. 60. The kit described in item 59, further comprising peptides / polypeptides for use in therapy, prevention, and / or diagnosis.

[0084] This disclosure includes combinations of the described embodiments and preferred features, unless the combination is clearly unacceptable or expressly avoided. Section headings used in this specification are for organizational purposes only and should not be construed as limiting the scope of the subject matter. Herein, aspects and embodiments of this disclosure are illustrated by reference to the accompanying drawings as an example. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0085] Throughout this specification, including the subsequent claims, unless otherwise required by context, the word “comprise” and its variations, e.g., “comprises” and “comprising,” are understood to mean that they encompass the presented integer or process or group of integers or processes, but not that they exclude any other integers and processes or groups of integers and processes. As used herein, the term “prevent” may also mean “reduce.” For example, in various aspects of the present invention, “preventing” the degradation of polysorbate, for example, means “reducing” the degradation of polysorbate, for example.

[0086] When used in the specification and the appended claims, the singular forms "a," "an," and "the" should be noted to include multiple references unless the context clearly indicates otherwise. Ranges may be expressed herein as "about" one specific value and / or "about" the other specific value. When such ranges are expressed, alternative embodiments include one specific value and / or the other specific value. Similarly, when values ​​are expressed as approximations, it is understood that by using the aforementioned "about," a particular value forms an alternative embodiment.

[0087] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length ranging from about 2 to about 50 amino acids. A "polypeptide" contains two or more peptide bonds and contains three or more amino acids. Values ​​may be expressed herein as "approximately" a specific value. Similarly, ranges may be expressed herein as "approximately" from one specific value and / or "approximately" to another specific value. The term "approximately" is arbitrary with respect to numbers and means, for example, + / - 10%. For example, when referring to "approximately 10%", it is interpreted as 9% to 11%. Where "approximately" is listed herein, the values ​​that follow are also considered in detail. For example, when referring to "approximately 10%", 10% is also considered in detail. [Examples]

[0088] (Example 1) Oxidative stress stability test in the presence of citrate 1.1 Test Setup Sodium citrate dihydrate was used in the preparation of the citrate-containing formulation. The sample solution was filtered through a 0.22 μm porosity filter cartridge (Sterivex-GV, Millipore), and a fixed volume of 50 mL of sterile bulk formulation was transferred into a 50R borosilicate glass vial. To enhance oxidative stress during stability testing, hydrogen peroxide (H2O2) was added to PS20 samples stored at 40°C. The final adjusted H2O2 concentration was 1000 ppm (29 mM) in the 8-week test and 1 ppm (29 μM) in the long-term tests (extended to 12 and 24 months). Vials were sealed with Teflon and silicone-treated bromobutyl D777-1 stoppers and then sealed with aluminum crimp caps. Samples were stored at 40°C for up to 12 months initially and up to 24 months, and analyzed as follows. Containers selected for PS20-related analysis were randomly selected. The tested samples contained 0.9% (w / v) NaCl (physiological saline) with a citrate-to-PS20 molar ratio ranging from 0.06 to 15.4 in 0.2 mg / ml PS20. 50 ml of each formulation was filled into 50R glass vials and sealed with rubber stoppers. The polysorbate content of samples at specific time points was analyzed using a fluorescence micelle assay (FMA).

[0089] In the oxidative stress stability test, to identify the optimal citrate-to-PS20 molar ratio, H2O2 was added to the sample until the final concentration reached 29 mM (1000 ppm) H2O2, and the sample was stored at 40°C for up to 8 weeks. Long-term stability test samples with a citrate-to-PS20 molar ratio of 3 at a final H2O2 concentration of 29 μM (1 ppm) were initially stored at 40°C for up to 12 months, and then extended to 24 months at 40°C. The polysorbate content of the samples at specific time points was analyzed using fluorescence micelle assay (FMA). In the oxidative stress stability test, to identify the optimal citrate to PS 80 molar ratio, H2O2 was added to the sample until the final concentration reached 29 mM (1000 ppm) H2O2, and the sample was stored at 40°C for up to 8 weeks.

[0090] 1.2 Investigation of the effect of various molar ratios of citric acid to PS20 in the formulation on the decomposition of PS20 Oxidative stress stability tests were conducted to investigate the degradation of PS20 in aqueous formulations in the absence and presence of citrate at various citrate-to-PS20 molar ratios. The formulation was 0.2 mg / ml. -1 The solution contained PS20 (equivalent to 0.02% w / v) and 0.9% (w / v) NaCl, and oxidative stress was induced by adding 29 mM H2O2. Table 1 outlines the composition of each tested formulation. Molar concentrations are calculated based on the molecular weight (Mr) of each component (PS20 Mr = 1227 g / mol, sodium citrate dihydrate Mr = 294.10 g / mol). [Table 1] The results are shown in Figure 2. As can be seen in Figure 2, in the absence of citrate, PS20 decomposes rapidly, with 50% of PS20 decomposing after 2 weeks of storage at 40°C. After 6 weeks of storage at 40°C, the intact PS20 content decreases to less than 20% of the initial value.

[0091] At a citrate-to-PS20 molar ratio of 0.06, the presence of citrate reduces the degradation kinetics of PS20. However, even when the citrate-to-PS20 molar ratio is increased to 0.31, approximately 50% of the PS20 is still degraded after 8 weeks (at 40°C). In the examples described herein, in addition to 29 μM H2O2, a storage temperature of 40°C was selected as an additional stress factor, which was assumed to accelerate degradation. If stability is demonstrated during storage at 40°C for a specific period of time, at least the same stability (over the same period of time) can be expected at lower temperatures, e.g., around 25°C. Typically, stability is improved (i.e., over a longer period of time) at lower temperatures, e.g., around 25°C. These results differ from the data reported by Doyle et al. (2019)(38), who reported that a citrate concentration of 62.5 μM, corresponding to a citrate-to-PS80 molar ratio of 0.4, was sufficient to prevent oxidation of polysorbate 80 (PS80) in histidine (10 mM) preparations exposed to stainless steel. To reduce the degradation of PS20, this study found that a citrate-to-PS20 molar ratio of at least 0.61 was required (Figure 2).

[0092] Since histidine has been reported to protect polysorbates from oxidation induced by (2,2'-azobis(2-amidinopropane) hydrochloride) (APPH), a model of reactive oxygen species (40), the presence of histidine buffers in formulations examined by Doyle et al. (2019) (38) may interfere with or block the antioxidant effect of citrate. As described above in this specification, both PS80 and PS20 are polysorbates, but the chemical compositions of these two surfactants differ (17). Some reports have also described the differences in the degradation sensitivities of the two polysorbates to specific stress conditions (4, 42, 43).

[0093] Given the structural differences between PS80 and PS20 and the presence of histidine buffers in the formulations as reported by Doyle et al. (2019)(38), it is impossible to rationally interpret or predict from the study by Doyle et al. (2019) the specific effect of citrate on the oxidation of PS20. This study focuses on the stability of PS20 in citrate-containing formulations in the absence of histidine. The presence of small amounts of citrate can impair protein stability (16, 18, 19, 44-46). Gopalrathnam et al. (2018) concluded that the use of citrate had a negative effect on product stability, stating that "in the LY2951742 antibody formulation, sodium citrate buffering was not considered a viable option because its stability performance was below optimal" (47). Therefore, careful evaluation of the citrate content used to stabilize polysorbates requires ensuring that the stability of any polysorbates present in the formulation is not adversely affected.

[0094] In this study, we found that a citrate-to-PS20 molar ratio of 0.31, which closely corresponds to the conditions tested by Doyle et al. (2019)(38), did not prevent the oxidation of PS20, as 45% of PS20 decomposed after 8 weeks at 40°C. The inventors' citrate titration test surprisingly reveals the optimal citrate-to-PS20 molar ratio required to reduce the degradation of PS20. A citrate-to-PS20 molar ratio of at least approximately 1.5 to 3 is required to reduce PS20 degradation. That is, the minimum citrate-to-PS20 molar ratio that reduces degradation is in the range of approximately 1.5 to 3. Such data indicates that approximately 10 times more citrate is required to stabilize PS20 compared to PS80 as proposed by Doyle et al. (2019)(38). As shown in Figure 2, a citrate-to-PS20 molar ratio in the range of approximately 1.5 to 6 results in improved PS20 stabilization over a period of up to 2 months (at least a twofold improvement compared to citrate-free formulations). Therefore, the optimal citrate-to-PS20 molar ratio is in the range of approximately 1.5 to 6 or even 1.5 to 7.

[0095] At citrate-to-PS20 molar ratios greater than approximately 7, for example, the ratio of 15.4 shown in Figure 2, PS20 is slightly more decomposed. PS20 exhibited good stability over the test period (0.5 months, 1 month, 1.5 months, and 2 months), and in formulations using citrate-to-PS20 molar ratios ranging from approximately 1.5 to approximately 6, PS20 consistently degraded at a low rate. Within this range, a partial range of citrate-to-PS20 molar ratios from approximately 3 to approximately 6 resulted in a clear improvement of more than four times in stability at all time points compared to citrate-free formulations (Figure 2).

[0096] The highest PS20 stability was observed in formulations using citrate-to-PS20 molar ratios of 3.07 or 4.61, showing less than 20% PS20 degradation over 2 months at 40°C in the presence of 29 mM H2O2, with no S20 degradation for up to 0.5 months. Considering concerns that high concentrations of PS20 may affect polypeptide stability, we selected a citrate-to-PS20 molar ratio of approximately 3 (0.5 mM citrate) and conducted further investigations.

[0097] 1.3 Investigation of the effect of various molar ratios of citrate to PS80 in the formulation on the degradation of PS80 Oxidative stress stability tests were conducted according to the procedure described in Example 1.2 to investigate the degradation of PS80 in aqueous formulations in the absence and presence of citrate at various citrate-to-PS80 molar ratios. The formulation was 0.2 mg / ml. -1 The solution contained PS80 (equivalent to 0.02% w / v) and 0.9% (w / v) NaCl, and oxidative stress was induced by adding 29 mM H2O2. Table 2 outlines the composition of each tested formulation. Molar concentrations are calculated based on the molecular weight (Mr) of each component (PS80 Mr = 1310 g / mol, sodium citrate dihydrate Mr = 294.10 g / mol). [Table 2] The results are shown in Figure 5. In the absence of citrate, PS80 decomposes rapidly, with 100% of PS80 being decomposed after 4 weeks of storage at 40°C. At a citrate-to-PS80 molar ratio of 0.33, the presence of citrate reduces the degradation kinetics of PS80. However, after 8 weeks (at 40°C), approximately 70% of the PS80 is degraded.

[0098] In the examples described herein, in addition to 29 μM H2O2, a storage temperature of 40°C was selected as a further stress factor, which was assumed to accelerate degradation. If stability is demonstrated during storage at 40°C for a specific period of time, at least the same stability (over the same period of time) can be expected at lower temperatures, e.g., around 25°C. Typically, stability is improved (i.e., over a longer period of time) at lower temperatures, e.g., around 25°C. These results differ from the data reported by Doyle et al. (2019)(38), who reported that a citrate concentration of 62.5 μM, corresponding to a citrate-to-PS80 molar ratio of 0.4, was sufficient to prevent oxidation of polysorbate 80 (PS80) in histidine (10 mM) preparations exposed to stainless steel. This study found that a citrate-to-PS80 molar ratio of at least 0.66 was required to significantly reduce the degradation of PS80 (Figure 5).

[0099] In this study, we found that at a citrate-to-PS80 molar ratio of 0.33, which is roughly equivalent to the conditions tested by Doyle et al. (2019)(38), 70% of the PS80 decomposed after 8 weeks at 40°C, indicating that the citrate-to-PS80 ratio did not prevent oxidation of PS80. The inventor's citrate titration test surprisingly reveals the optimal citrate-to-PS80 molar ratio required to reduce the degradation of PS80. A citrate-to-PS80 molar ratio of at least approximately 1.6 to 3.2 is required to reduce PS80 degradation. That is, the minimum citrate-to-PS80 molar ratio that reduces degradation is in the range of approximately 1.6 to 3.2. Such data indicates that approximately four times more citrate is required to stabilize PS80 compared to the amount proposed by Doyle et al. (2019)(38). As shown in Figure 5, a citrate-to-PS80 molar ratio in the range of approximately 1.6 to 6.5 results in improved PS80 stabilization over a period of up to two months (at least a twofold improvement compared to citrate-free formulations). Therefore, the optimal molar ratio of citrate to PS80 is in the range of approximately 1.6 to 6.5 or even 1.6 to 7.

[0100] Furthermore, this data indicates that at citrate-to-PS80 molar ratios greater than approximately 7, for example, the ratio of 16.4 shown in Figure 5, PS80 is slightly more degraded. Doyle et al. (2019) could not identify this trend. PS80 exhibited good stability over the test period (1 month, 1.5 months, and 2 months), and in formulations using citrate-to-PS80 molar ratios ranging from approximately 1.6 to approximately 6.5, PS80 consistently degraded at a low rate. Within this range, over 65% of PS80 was stabilized by a partial range of citrate-to-PS80 molar ratios from approximately 3 to approximately 6, while the citrate-free sample contained 0% PS80 (Figure 5).

[0101] The highest PS80 stability was observed in formulations using citrate-to-PS80 molar ratios of 3.28 or 4.91, showing less than 33% PS80 degradation over 2 months at 40°C in the presence of 29 mM H2O2, and approximately 20% PS80 degradation over a maximum of 1 month. Formulations using a citrate-to-PS80 molar ratio of 6.55 also showed less than 33% PS80 degradation observed over 2 months. These results indicate that the stabilization of PS80 in sodium chloride-containing formulations and even in the absence of histidine buffers is achieved at a citrate-to-PS80 molar ratio of approximately 1.6 to 7, with the optimal range being approximately 3 to 6.

[0102] (Example 2) Citrate provides long-term protection against oxidative degradation of PS20. 2.1 Long-term stability test of PS20 formulations containing citrate As shown in Figure 2, a citrate-to-PS20 molar ratio of approximately 3 is the minimum amount required to reduce PS20 degradation, therefore, 0.2 mg / ml in MilliQ water is needed. -1 Stability tests were extended in the presence and absence of this citrate-to-PS20 molar ratio using PS20 and 0.9% (w / v) NaCl. Furthermore, the presence of sodium chloride on the stability of PS20 was evaluated. Therefore, long-term oxidative stability tests were conducted to monitor whether a citrate-to-PS20 molar ratio of 3 was sufficient to stabilize PS20 for up to 12 months and even up to 24 months.

[0103] Figures 3A and 3B demonstrate the results of the long-term stability test. Both formulations, in the absence of citrate, showed a PS20 content of 0.025 mg / ml after 3 months at 40°C. -1 It is clear that the PS has dropped to below 20. Two formulations prepared in the presence of citrate showed stability of PS20 during storage at 40°C for up to 12 months (Figure 3A). Therefore, a citrate-to-PS20 molar ratio of approximately 3 is efficient in preventing PS20 degradation for at least 12 months. A slight decrease of about 10% was observed, which the inventors predict is due to different degradation pathways, as reported by Dwivedi et al. (17). The presence of sodium chloride does not have a significant effect on the stability of PS20.

[0104] As shown in Figure 3B, significant stabilization of PS20 is maintained even after storage at 40°C for up to 24 months. The concentration of PS20 in citrate-containing samples after 24 months of storage at 40°C was 0.1–0.15 mg / ml. -1 While it is well maintained above a certain level, in the absence of citrate, detectable levels of PS20 are practically nonexistent.

[0105] 2.2 Investigation of oxidative degradation markers of PS20 To gain further insight into the degradation mechanism of PS20 in aqueous formulations, specific markers for oxidative degradation of PS20 were investigated. Table 3 shows key PS20 oxidation products that indicate oxidative degradation. [Table 3] Figure 4 demonstrates the levels of oxidation markers (POE5 Di, POE5 Mono, POE6 Di, POE6 Mono) detected in formulations containing or without PS20 and citrate at a citrate-to-PS20 molar ratio of 3.07. In the absence of citrate, formulations containing PS20 showed significant oxidation of PS20, as evidenced by the marker levels after 3 months at 40°C. Citrate-containing samples did not show an increase in oxidation marker levels, as illustrated in the graph showing levels after 6 months at 40°C. This finding is consistent with the results shown in Figure 3. The inventors identified various citrate-to-PS20 molar ratios that provide long-term protection of PS20 from oxidative degradation.

[0106] 2.3 Conclusion Stability experiments of the PS20-containing formulations described herein, including titration oxidation stability tests at 40°C, identified an optimal molar ratio of approximately 3–7 citrate to PS20. Within this range, the degradation of PS20 can be inhibited. Equivalent findings were obtained by the inventors when formulations containing another polysorbate (polysorbate 80) were tested. The inventors identified an optimal molar ratio of approximately 3–7 citrate to PS that stabilizes the polysorbate in aqueous formulations. The inventors further evaluated the minimum molar ratio of citrate to PS20 of approximately 3 required for stabilization of PS20 in long-term oxidative stability tests of up to 12 months and even up to 24 months under extremely oxidative conditions (29 μM H2O2, 40°C). Under such oxidative conditions, 0.2 mg / ml in 0.9% (w / v) NaCl with a citrate-to-PS20 molar ratio of approximately 3 for up to 12 months at 40°C. -1 In formulations containing [the specified ingredient], no significant degradation of PS20 was observed, and even after 24 months under the same conditions, only minimal degradation of PS20 was observed.

[0107] This finding is particularly useful because the presence of citrate can induce the formation and / or gelation of protein particles during the processing and formulation of biologics (16, 18, 19, 44-46). Even at low citrate buffering concentrations down to 5 mM, reported case studies have described an increase in protein aggregation rates in citrate-containing formulations (46). A citrate to PS (e.g., PS20) molar ratio of approximately 3 is 0.2 mg / ml. -1 It can be converted to a citrate concentration in the range of 0.5 mM in formulations containing PS20, which is one-tenth of 5 mM. These results indicate that the stabilization of polysorbates (PS20, PS80, etc.) in sodium chloride-containing formulations and even in the absence of histidine buffers is achieved at a citrate-to-PS20 or citrate-to-PS80 molar ratio of approximately 1.5 to 7, with the optimal range being approximately 3 to 6.

[0108] (Example 3) Materials and methods used in Examples 1 and 2 3.1 Materials and Reagents Acetonitrile (ACN) for liquid chromatography, 35% (w / v) hydrogen peroxide (H2O2), ferrous ammonium(II) sulfate hexahydrate ((NH4)2Fe(SO4)×6H2O), and xylenol orange disodium salt (XODS) were obtained from Carl Roth GmbH (Karlsruhe, Germany). Ammonium formate (NH4HCO3), 30% (w / v) Brij-35, N-phenyl-1-naphthylamine (naphtylamin) (NPN), and iron chloride (FeCl2 and FeCl3) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Formic acid, sulfuric acid (H2SO4), and sodium chloride (NaCl) were obtained from Merck KgaA (Darmstadt, Germany). Sodium citrate dihydrate was obtained from Jungbunzlauer GmbH (Pernhofen, Australia). LC-MS grade methanol (MeOH) was purchased from Honeywell International Inc. (Charlotte, NC, USA), and trometamol (Tris) was obtained from Angus Chemie GmbH (Ibbenburen, Germany). High-purity grade polysorbate 20 (PS20 HP) was obtained from Croda (Arnhem, Netherlands). The pure PS20 HP material was stored under a nitrogen overlay at 2-8°C and protected from light. High-purity water was produced using the Milli-Q IQ 7000 system from Merck KgaA (Darmstadt, Germany) and will later be referred to as "water". 50 mL Fiolax® vials (Glass Type I) were purchased from Schott AG (Mainz, Germany). The 20mm B2 coated FluroTec (trademark) (D777-1) stoppers were purchased from West Pharmaceutical Services (Eschweiler, Germany). High-purity grade polysorbate 80 (PS80 HP) was obtained from Croda (Arnhem, Netherlands).

[0109] 3.2 Method Fluorescent micelle assay. The fluorescent micelle assay (FMA) was improved from Lipppold et al. (2017) (41). The concentration of PS20 was quantified by partitioning PS micelles using the hydrophobic fluorophore NPN (48). The PS20 HP concentration in the test solution was quantified using a Fluent automation workstation from Tecan Group AG (Mannedorf, Switzerland). Therefore, 240 μl of FMA buffer consisting of 5 μM NPN, 0.0015% (w / v) Brij-35, 150 mM NaCl, 5% (v / v) ACN, and 50 mM Tris was added to 10 μl of PS-containing sample at pH 8.0 and incubated at 35°C for 1 minute at 167 rpm. Four technical replicates (n=4) were measured. Sample fluorescence was detected at excitation wavelength 350 nm and emission wavelength 420 nm using a fluorescence plate reader (Infinite M200pro, Tecan Group AG, Mannedorf, Switzerland). PS20 concentrations were obtained using standard calibration samples of PS20 (0, 0.1, 0.3, 0.6 mg / ml). -1 The limit of detection (LOD) is 0.023 mg / ml. -1 The PS20 grade has a limit of quantification (LOQ) of 0.069 mg / ml. -1 The PS80 concentrations were obtained by using standard calibration samples of PS80 (0, 0.05, 0.2, 0.3 mg / ml). -1 The limit of quantification (LOQ) is 0.05 mg / ml. -1 That was the case.

[0110] Analysis of polysorbate 20 oxidation markers. Characterization of PS20 oxidation markers (see Table 3) was performed using a UPLC assay with a Qda mass detector, essentially following the method described in Birdsall et al., 'Quantitative Analysis of Polysorbate 20 / 80 in Protein-Based Biopharmaceuticals Using A One-Pot RPLC-MS Based Platform Method', Waters Corporation, GlaxoSmithKline; Application Note 720007249, May 2021.

[0111] (Example 4) Investigation of the effects of citrate-containing formulations as a diluent for proteins As described in Example 2, the citrate-containing formulation according to the present invention exhibits polysorbate stabilization. Considering studies (47) that report that high levels of citrate negatively affect the stability of polypeptides (e.g., antibodies) in formulations, the inventors further investigated the physical stability of protein samples prepared using the citrate-polysorbate formulation of the present invention as a diluent. The monoclonal antibody (mAb 1) was diluted in an aqueous solution of 0.9% (w / v) NaCl and 0.2 mg / mL polysorbate 20 in the absence of citrate (first diluent) or in the presence of 0.5 mM citrate (second diluent). By utilizing serial dilution with a specified diluent, the antibody concentration varied in each sample. The protein was diluted to produce the following samples: D0: undiluted, D01: 5.0 mg / mL, D02: 1.0 mg / mL, D03: 0.1 mg / mL, D04: 0.01 mg / mL, D05: 0.00001 mg / mL of mAb 1. Table 4 outlines the composition of each antibody preparation sample. Samples containing citrate had a citrate-to-PS20 molar ratio of 3.07. [Table 4] The solution was evaluated at the following time points: 0 hours, 9 hours, and 24 hours, by visual inspection, turbidity measurement, and measurement of the concentration of microparticles invisible to the naked eye. Two different storage conditions were tested: storage in the dark and storage under exposure.

[0112] Figures 6A and 6B demonstrate that the presence of low concentrations of citrate in antibody preparations does not significantly affect the turbidity of the antibody solution compared to the turbidity of samples without citrate, at various time points and even at high antibody concentrations. Visual inspection of the samples, conducted in accordance with the European Pharmacopoeia (Ph.Eur. 2.9.20), further confirmed that the presence of low concentrations of citrate does not induce the formation of visible particles in the samples (Figures 7A and 7B).

[0113] Microflow imaging (MFI) was used to determine the formation of particles invisible to the naked eye. All citrate-containing samples contained very low concentrations of microparticles ≥10 μm (Figure 8B) and microparticles ≥25 μm (Figure 9B), which were either lower than or comparable to the concentrations measured in the corresponding citrate-free samples (Figures 8A and 9A, respectively). These results demonstrate that the concentration of citrate and / or the molar ratio of citrate to PS according to the present invention prevents the degradation of PS without inducing an increase in turbidity, negatively affecting the photostability of proteins, or inducing particle formation over time in protein samples.

[0114] References JPEG2026511478000006.jpg70170 JPEG2026511478000007.jpg243167 JPEG2026511478000008.jpg228167 JPEG2026511478000009.jpg227167 JPEG2026511478000010.jpg204168

Claims

1. An aqueous formulation containing polysorbate, citrate, and sodium chloride.

2. The aqueous formulation according to claim 1, wherein the concentration of citrate in the aqueous formulation is about 50 μM to about 2.5 mM.

3. The aqueous formulation according to claim 1 or claim 2, wherein the concentration of citrate in the aqueous formulation is about 250 μM to about 1 mM.

4. The aqueous formulation according to any one of claims 1 to 3, wherein the concentration of polysorbate in the aqueous formulation is about 0.02% (w / v).

5. The aqueous formulation according to any one of claims 1 to 4, wherein the concentration of sodium chloride in the aqueous formulation is about 0.9% (w / v).

6. The aqueous formulation according to any one of claims 1 to 5, wherein the molar ratio of citrate to polysorbate in the aqueous formulation is about 1.5 to about 7.

7. The aqueous formulation according to any one of claims 1 to 6, wherein the molar ratio of citrate to polysorbate is about 3 to about 6.

8. An aqueous formulation according to any one of claims 1 to 7, wherein the molar ratio of citrate to polysorbate is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.

9.

9. The aqueous formulation according to any one of claims 1 to 8, wherein the polysorbate is polysorbate 20 or polysorbate 80, preferably polysorbate 20.

10. An aqueous formulation according to any one of claims 1 to 9, which does not contain histidine.

11. An aqueous formulation according to any one of claims 1 to 10, further comprising metal ions.

12. The aqueous formulation according to any one of claims 1 to 11, wherein the concentration of sodium chloride in the aqueous formulation is about 0.9% (w / v), the polysorbate is polysorbate 80, and the molar ratio of citrate to polysorbate in the aqueous formulation is about 3 to about 6.

13. An aqueous formulation according to any one of claims 1 to 12, comprising essentially polysorbate, citrate, water, and sodium chloride.

14. - Approximately 0.02% (w / v) of polysorbate, - Approximately 0.9% (w / v) sodium chloride, - Water, and - 0.25 mM to 1 mM citrate An aqueous formulation according to any one of claims 1 to 13, which is essentially derived from the above.

15. - Approximately 0.02% (w / v) of polysorbate, - Approximately 0.9% (w / v) sodium chloride, - Water, and - Approximately 0.5 mM citrate The aqueous formulation according to claim 14, which is essentially derived from the above.

16. An aqueous formulation containing polysorbate 20, citrate, and sodium chloride.

17. The aqueous formulation according to claim 16, wherein the concentration of citrate in the aqueous formulation is about 50 μM to about 2.5 mM.

18. The aqueous formulation according to claim 16 or claim 17, wherein the concentration of citrate in the aqueous formulation is about 250 μM to about 1 mM.

19. The aqueous formulation according to any one of claims 16 to 18, wherein the concentration of polysorbate 20 in the aqueous formulation is about 0.02% (w / v).

20. The aqueous formulation according to any one of claims 16 to 19, wherein the concentration of sodium chloride in the aqueous formulation is about 0.9% (w / v).

21. The aqueous formulation according to any one of claims 16 to 20, wherein the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 1.5 to about 7.

22. The aqueous formulation according to any one of claims 16 to 21, wherein the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3 to about 6.

23. The aqueous formulation according to any one of claims 16 to 22, wherein the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.

9.

24. An aqueous formulation according to any one of claims 16 to 23, which does not contain histidine.

25. An aqueous formulation according to any one of claims 16 to 24, further comprising metal ions.

26. The aqueous formulation according to any one of claims 16 to 25, wherein the concentration of sodium chloride in the aqueous formulation is about 0.9% (w / v), and the molar ratio of citrate to polysorbate 20 in the aqueous formulation is about 3 to about 6.

27. An aqueous formulation according to any one of claims 16 to 26, comprising essentially polysorbate 20, citrate, water, and sodium chloride.

28. - Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - Water, and - 0.25 mM to 1 mM citrate An aqueous formulation according to any one of claims 16 to 27, which is essentially derived from the above.

29. - Approximately 0.02% (w / v) of polysorbate 20 (PS20), - Approximately 0.9% (w / v) sodium chloride, - Water, and - Approximately 0.5 mM citrate The aqueous formulation according to claim 28, which is essentially derived from the above.

30. An aqueous formulation according to any one of claims 1 to 29, suitable for intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal administration.

31. An aqueous formulation according to any one of claims 1 to 30, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

32. An aqueous formulation according to any one of claims 1 to 31, provided in a container including a glass container, a stainless steel container, or a plastic container.

33. An aqueous formulation according to any one of claims 1 to 32, further comprising a peptide / polypeptide for use in therapy, prevention, and / or diagnosis.

34. The aqueous formulation according to claim 33, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

35. A method for preventing the decomposition of polysorbate in an aqueous formulation, comprising the step of adding citrate in a final molar ratio of citrate to polysorbate of about 1.5 to about 7.

36. The use of citrate to prevent the decomposition of polysorbate in an aqueous formulation, wherein the final molar ratio of citrate to polysorbate is approximately 1.5 to approximately 7.

37. The method according to claim 35 or the use according to claim 36, wherein the polysorbate is polysorbate 20 or polysorbate 80, preferably polysorbate 20.

38. The method according to claim 35 or 37, or the use according to claim 36 or 37, wherein the aqueous formulation contains sodium chloride.

39. The method according to any one of claims 35, 37, and 38 or the use according to any one of claims 36, 37, and 38, wherein the aqueous formulation is the aqueous formulation according to any one of claims 1 to 15.

40. The method according to any one of claims 35 and 37-39, or the use according to any one of claims 36-39, wherein the decomposition of the polysorbate includes oxidation of the polysorbate.

41. The method according to any one of claims 35 and 37-40, or the use according to any one of claims 36-40, wherein the decomposition of the polysorbate includes the decomposition of the polysorbate in the presence of metal ions.

42. The method according to any one of claims 35 and 37-41, or the use according to any one of claims 36-41, wherein the degradation of the polysorbate comprises the degradation of the polysorbate in the presence of a peptide / polypeptide for use in therapy, prevention, and / or diagnosis.

43. The method or use according to claim 42, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

44. The method according to any one of claims 35 and 37-43 or the use according to any one of claims 36-43, wherein the aqueous formulation is provided in a container including a glass container, a stainless steel container, or a plastic container.

45. The method according to any one of claims 35 and 37-44, or the use according to any one of claims 36-44, wherein the molar ratio of citrate to polysorbate is about 3 to about 6.

46. The method according to any one of claims 35 and 37-45, or the use according to any one of claims 36-45, wherein the molar ratio of citrate to polysorbate is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.

9.

47. The method according to any one of claims 35 and 37 to 46, or the use according to any one of claims 36 to 46, wherein the aqueous formulation contains about 0.02% (w / v) of polysorbate.

48. A method for preventing the decomposition of polysorbate 20 in an aqueous formulation, comprising the step of adding citrate in a final molar ratio of citrate to polysorbate 20 of about 1.5 to about 7.

49. The use of citrate to prevent the decomposition of polysorbate 20 in an aqueous formulation, where the final molar ratio of citrate to polysorbate 20 is approximately 1.5 to approximately 7.

50. The method according to claim 48 or the use according to claim 49, wherein the aqueous formulation contains sodium chloride.

51. The method according to claim 48 or 50 or the use according to claim 49 or 50, wherein the aqueous formulation is the aqueous formulation according to any one of claims 16 to 34.

52. The method according to any one of claims 48 and 50-51, or the use according to any one of claims 49-51, wherein the decomposition of polysorbate 20 includes oxidation of polysorbate 20.

53. The method according to any one of claims 48 and 50-52, or the use according to any one of claims 49-52, wherein the decomposition of polysorbate 20 includes the decomposition of polysorbate 20 in the presence of metal ions.

54. The method according to any one of claims 48 and 50-53 or the use according to any one of claims 49-53, wherein the degradation of polysorbate 20 comprises the degradation of polysorbate 20 in the presence of peptides / polypeptides for use in therapy, prevention, and / or diagnosis.

55. The method or use according to claim 54, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

56. The method according to any one of claims 48 and 50-55 or the use according to any one of claims 49-55, wherein the aqueous formulation is provided in a container including a glass container, a stainless steel container, or a plastic container.

57. The method according to any one of claims 48 and 50-56, or the use according to any one of claims 49-56, wherein the molar ratio of citrate to polysorbate 20 is about 3 to about 6.

58. The method according to any one of claims 48 and 50-57, or the use according to any one of claims 49-57, wherein the molar ratio of citrate to polysorbate 20 is about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.

9.

59. The method according to any one of claims 48 and 50-58 or the use according to any one of claims 49-58, wherein the aqueous formulation contains about 0.02% (w / v) of polysorbate 20.

60. Use of the aqueous formulation according to any one of claims 1 to 34 as a diluent for the composition.

61. The use according to claim 60, wherein the composition comprises a peptide / polypeptide for use in therapy, prevention, and / or diagnosis.

62. The use according to claim 61, wherein the peptide / polypeptide for use in therapy, prevention, and / or diagnosis is selected from antigen-binding peptides / polypeptides, antigen-binding peptide / polypeptide complexes, antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

63. A method for preparing a composition, comprising the step of contacting a peptide / polypeptide for use in therapy, prevention, and / or diagnosis with an aqueous formulation according to any one of claims 1 to 34.

64. The method according to claim 63, wherein the composition is a pharmaceutical composition for administration.

65. The method according to claim 64, wherein the administration is intravenous, parenteral, systemic, intracavitary, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subconjunctival, intravitreous, intratumoral, subcutaneous, intradermal, oral, or transdermal.

66. A composition obtained or obtainable by the method described in any one of claims 63 to 65.

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