Compositions and methods for reducing oxidation of IGF-1 / IGFBPs - Patent application

Abstract

The present invention provides compositions and methods for reducing oxidation of a complex comprising insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3), inter alia, for treating diseases and complications in low birth weight infants. In some embodiments, the method comprises adding an antioxidant. In some embodiments, the antioxidant is methionine or sodium thiosulfate. In some aspects, the compositions provided are stable, pure and potent compositions comprising IGF-1 and IGFBP-3. In some aspects, the formulations are lyophilized.

Description

[Technical field]

[0001] The present disclosure relates to stable formulations of mecasermin rinfavert (rhIGF-1 / rhIGFBP-3), in which oxidation of one or more species is minimized, e.g., upon storage at 2-8°C and / or 25°C, methods of making said formulations, and uses thereof in therapy, particularly in the treatment of infants, e.g., premature infants. [Background technology]

[0002] Mecasermin rinfavert is a complex of recombinant human insulin-like growth factor-1 (rhIGF-1) and recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) used to treat diseases and complications in low birth weight infants, such as intraventricular hemorrhage, bronchopulmonary dysplasia, and / or chronic lung disease in low birth weight infants.

[0003] For example, there is a need for formulations specifically optimized for greater stability of therapeutic protein products at 2-8° C., 25° C. or 40° C. during storage for about 3-6 months or longer.

[0004] Extremely dilute formulations are needed to treat infants, especially premature infants. Mecasermin rinfavert is typically administered to premature infants by continuous intravenous infusion. Surprisingly, the inventors have demonstrated that in dilute formulations, the complex oxidizes more rapidly. Summary of the Invention

[0005] The present invention provides, in part, compositions and methods for reducing oxidation of formulations that include a protein complex that includes insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3). Without wishing to be bound by any particular theory, it is contemplated that methods for reducing oxidative species (e.g., addition of antioxidants, lyophilization, and / or disposable bags) provide improved formulations with greater potency and increased stability.

[0006] The disclosure is summarized in the following numbered paragraphs: 1. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and an insulin-like growth factor binding protein (e.g., IGFBP-3), e.g., in a 1:1 molar ratio, wherein the composition contains an antioxidant, and oxidized IGF-1 species does not increase by more than 50% after 3 months of storage at a temperature of about 25°C.

[0007] 2. The composition of paragraph 1, wherein oxidized IGF-1 species does not increase by more than 25% after 3 months of storage at a temperature of about 25°C.

[0008] 3. The composition of paragraphs 1 or 2, wherein oxidized IGF-1 species does not increase by more than 10% after 3 months of storage at a temperature of about 25°C.

[0009] 4. The composition of paragraph 1, wherein oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature of about 25°C.

[0010] 5. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and an insulin-like growth factor binding protein (e.g., IGFBP-3), e.g., in a 1:1 molar ratio, wherein the composition contains an antioxidant, and the % of oxidized IGF-1 species does not increase by more than 20% after 3 months of storage at a temperature of 2-8°C.

[0011] 6. The composition of paragraph 5, wherein oxidized IGF-1 species does not increase by more than 10% after 3 months of storage at a temperature of 2-8°C.

[0012] 7. The composition of paragraph 6, wherein oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature of 2-8°C.

[0013] 8. The composition of any one of the preceding paragraphs, wherein the antioxidant is methionine.

[0014] 9. The composition of any one of the preceding paragraphs, wherein the composition comprises methionine at a concentration between 0.2 mM and 50 mM.

[0015] 10. The composition of paragraph 9, wherein the composition comprises methionine at a concentration of 0.5 mM.

[0016] 11. The composition of paragraph 9, wherein the composition comprises methionine at a concentration of 5 mM.

[0017] 12. The composition of any one of paragraphs 1-7, wherein the antioxidant is sodium thiosulfate.

[0018] 13. The composition of any one of the preceding paragraphs, wherein the composition comprises sodium thiosulfate in a concentration between 0.2 mM and 50 mM.

[0019] 14. The composition of paragraph 13, wherein the composition comprises sodium thiosulfate at a concentration of 0.5 mM.

[0020] 15. The composition of paragraph 13, wherein the composition comprises sodium thiosulfate at a concentration of 5 mM.

[0021] 16. The composition of any one of the preceding paragraphs, wherein less than 1% of the IGF-1 is present as an oxidized species in the protein complex upon 3 months of storage.

[0022] 17. The composition of any one of the preceding paragraphs, wherein less than 0.6% of the IGF-1 is present as an oxidized species in the protein complex upon 3 months of storage.

[0023] 18. The composition of any one of the preceding paragraphs, wherein the amount of oxidized species is determined by RP-UPLC or SEC-HPLC.

[0024] 19. The composition of any one of the preceding paragraphs, wherein the IGFBP-3 contains less than 5% trisulfide variants.

[0025] 20. The composition of paragraph 19, wherein the IGFBP-3 contains less than 2% trisulfide variants.

[0026] 21. The composition of paragraph 19, wherein the IGFBP-3 contains less than 1% trisulfide variants.

[0027] 22. The composition of paragraph 19, wherein the IGFBP-3 contains less than 0.5% trisulfide variants.

[0028] 23. A method for producing an insulin-like growth factor 1 (IGF-1) protein complex, comprising the steps of: providing a composition comprising recombinant IGF-1; adding an antioxidant to the composition; and purifying IGF-1 from the composition.

[0029] 24. The method of paragraph 23, wherein the antioxidant is methionine.

[0030] 25. The method of paragraph 23, wherein methionine is added at a concentration of 0.2 mM to 10 mM.

[0031] 26. The method of paragraph 23, wherein methionine is added at a concentration of 0.5 mM.

[0032] 27. The method of paragraph 23, wherein methionine is added at a concentration of 5 mM.

[0033] 28. The method of paragraph 23, wherein the antioxidant is sodium thiosulfate.

[0034] 29. The method of paragraph 28, wherein sodium thiosulfate is added at a concentration of 0.2 mM to 10 mM.

[0035] 30. The method of paragraph 28, wherein sodium thiosulfate is added at a concentration of 0.5 mM.

[0036] 31. The method of paragraph 28, wherein sodium thiosulfate is added at a concentration of 5 mM.

[0037] 32. The method of any one of paragraphs 23-31, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 50% after 3 months of storage at a temperature of about 25°C.

[0038] 33. The method of any one of paragraphs 23-31, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 20% after 3 months of storage at a temperature of about 25°C.

[0039] 34. The method of any one of paragraphs 23-31, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 10% after 3 months of storage at a temperature of about 25°C.

[0040] 35. The method of any one of paragraphs 23-31, wherein the oxidized IGF-1 species in the purified IGF-1 composition is not substantially increased after 3 months of storage at a temperature of about 25°C.

[0041] 36. The method of any one of paragraphs 23-31, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 20% after 3 months of storage at a temperature of 2-8°C.

[0042] 37. The method of any one of paragraphs 23-31, wherein the amount of oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 10% after 3 months of storage at a temperature of 2-8°C.

[0043] 38. The method of any one of paragraphs 23-31, wherein the amount of oxidized IGF-1 species in the purified IGF-1 composition does not increase substantially after 3 months of storage at a temperature of 2-8°C.

[0044] 39. The method of any one of paragraphs 23-31, wherein the purified IGF-1 protein has less than 0.6% oxidized IGF-1 species.

[0045] 40. The method of any one of paragraphs 23 to 39, wherein the recombinant IGF-1 protein is expressed in Escherichia coli.

[0046] 41. The method of any one of paragraphs 23 to 40, wherein the recombinant IGF-1 protein is purified from inclusion bodies.

[0047] 42. The method of any one of paragraphs 23-41, wherein the method further comprises adding insulin-like growth factor binding protein 3 (IGFBP-3) to the IGF-1 protein to form a protein complex.

[0048] 43. The method of paragraph 42, wherein the protein complex comprises IGF-1 and IGFBP-3 in a 1:1 molar ratio.

[0049] 44. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3), produced by the method of any one of paragraphs 23 to 43.

[0050] 45. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3), for example in a 1:1 molar ratio, the composition comprising a bulking agent, a buffer and a surfactant.

[0051] 46. ​​The composition of paragraph 45, wherein the bulking agent is sucrose or trehalose.

[0052] 47. The composition of paragraph 46, wherein the bulking agent is trehalose.

[0053] 48. The composition of paragraph 45, wherein the buffer is histidine or phosphate.

[0054] 49. The composition of paragraph 48, wherein the buffer has a pH of 5.5-6.5.

[0055] Improved stability is achieved during storage at about 25° C. (e.g., about 23° C. to about 27° C.), or during storage at about 40° C. (e.g., about 38° C. to about 42° C.), for example, for about 3-6 months.

[0056] In one embodiment, provided herein are improved IGF-1 / IGFBP-3 formulations that include an antioxidant (eg, methionine and / or sodium thiosulfate).

[0057] In other embodiments, the oxidized species in the formulation are reduced by lyophilization.

[0058] In other embodiments, the concentrated protein is formulated in a vial for dilution prior to use.

[0059] In one embodiment, the disclosure provides a lyophilized formulation that includes an antioxidant (e.g., methionine and / or sodium thiosulfate).

[0060] In one embodiment, the disclosure provides a high concentration formulation that includes an antioxidant (e.g., methionine and / or sodium thiosulfate).

[0061] In other embodiments, reduced accumulation of oxidative species and improved stability is achieved through the use of contact surfaces other than stainless steel, e.g., disposable bags, during compounding of the complex with the formulation solution; including formulations containing antioxidants (e.g., methionine and / or sodium thiosulfate) and / or high concentration formulations.

[0062] In summary, the present invention provides improved formulations of IGF-1 / IGFBP-3 that have the advantage, inter alia, of improved stability during storage, providing a safe and effective product for administration to neonates in the treatment of diseases of low birth weight infants.

[0063] In one aspect, provided herein is a composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3), e.g., in a 1:1 molar ratio, wherein the composition comprises an antioxidant, and the amount of oxidized IGF-1 species does not increase by more than 50%, e.g., does not increase by more than 45%; e.g., does not increase by more than 40%, particularly does not increase by more than 35%; more particularly does not increase by more than 30% after 3 months of storage at a temperature of about 25° C. In some embodiments, after 3 months of storage at a temperature of about 25° C., the amount of oxidized IGF-1 species does not increase by more than: 25%; 20%; 15%; 10%; 5%; 4%; 3%; 2%; or 1%. * .

[0064] * Each of these values ​​represents an individual disclosure contained in the priority application and may be used independently as a basis for amending the claims.

[0065] In some embodiments, the amount of oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature of about 25°C.

[0066] Molar ratios of IGF-1:IGFBP-3 that can be used in the formulations of the present disclosure include 1.5-0.5 IGF-1:1 IGFBP-3.

[0067] In one embodiment, provided herein is a composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, wherein the amount of oxidized IGF-1 species does not increase by more than 20% after 3 months of storage at a temperature of 2-8° C.

[0068] In some embodiments, the amount of oxidized IGF-1 species does not increase by more than 15%, such as not increasing by more than 10%, such as not increasing by more than 5%, particularly not increasing by more than 4%, and more particularly not increasing by more than 3% after 3 months of storage at a temperature of 2-8° C. In some embodiments, the amount of oxidized IGF-1 species does not increase by more than 2% after 3 months of storage at a temperature of 2-8° C. In some embodiments, the amount of oxidized IGF-1 species does not increase by more than 1% after 3 months of storage at a temperature of 2-8° C. In some embodiments, the amount of oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature of 2-8° C.

[0069] In one embodiment, provided herein are compositions or methods according to the disclosure, wherein the antioxidant is methionine.

[0070] In one embodiment, compositions or methods according to the present disclosure are provided herein that include methionine (including the addition of an antioxidant such as methionine) at a concentration in the range (e.g., between) 0.2 mM to 15 mM, such as 0.2 mM to 10 mM. In one embodiment, methionine is at a concentration of 0.2 mM; 0.3 mM; 0.4 mM; 0.5 mM; 0.6 mM; 0.7 mM; 0.8 mM; 0.9 mM; 1 mM; 2 mM; 3 mM; 4 mM; 5 mM; 6 mM; 7 mM; 8 mM; or 9 mM. * In one embodiment, a composition or method according to the present disclosure is provided herein that includes methionine (including adding an antioxidant such as methionine) at a concentration of 10 mM.

[0071] In one embodiment, provided herein is a composition or method according to the present disclosure, wherein the antioxidant is sodium thiosulfate.

[0072] In one embodiment, provided herein is a composition or method according to the disclosure, wherein the composition comprises (including the addition of) sodium thiosulfate at a concentration in the range (e.g., between) 0.2 mM and 15 mM, such as 0.2 mM and 10 mM.

[0073] In one embodiment, a composition or method according to the present disclosure comprises sodium thiosulfate at 0.2 mM; 0.3 mM; 0.4 mM; 0.5 mM; 0.6 mM; 0.7 mM; 0.8 mM; 0.9 mM; 1 mM; 2 mM; 3 mM; 4 mM; 5 mM; 6 mM; 7 mM; 8 mM; or 9 mM. * In one embodiment, a composition is provided herein comprising sodium thiosulfate at a concentration of 10 mM. In one embodiment, a composition is provided herein comprising sodium thiosulfate at a concentration of 10 mM.

[0074] In one embodiment, provided herein is a composition, wherein less than 5%, such as less than 4%, such as less than 3%, particularly less than 2%, particularly less than 1% of IGF-1 is present as oxidized species in the protein complex upon storage for 3 months.

[0075] In one embodiment, compositions are provided herein in which less than 0.9% of IGF-1 is present as oxidized species in protein complexes after 3 months of storage, for example, less than 0.8% of IGF-1 is present as oxidized species, for example, less than 0.7% of IGF-1 is present as oxidized species, particularly less than 0.6% of IGF-1 is present as oxidized species in protein complexes after 3 months of storage, more particularly less than 0.5% of IGF-1 is present as oxidized species.In one embodiment, compositions are provided herein in which less than 0.4% of IGF-1 is present as oxidized species in protein complexes after 3 months of storage.In one embodiment, compositions are provided herein in which less than 0.3% of IGF-1 is present as oxidized species in protein complexes after 3 months of storage.In one embodiment, compositions are provided herein in which less than 0.2% of IGF-1 is present as oxidized species in protein complexes after 3 months of storage. In one embodiment, provided herein is a composition, wherein less than 0.1% of the IGF-1 is present as an oxidized species in the protein complex upon 3 months of storage.

[0076] In one embodiment, compositions provided herein, wherein the amount of oxidized species is determined by reverse phase-ultra performance liquid chromatography (RP-UPLC).

[0077] In one embodiment, compositions provided herein, wherein the amount of oxidized species is determined by size exclusion chromatography-HPLC separation mode (SEC-HPLC).

[0078] In one embodiment, provided herein is a composition wherein the IGFBP-3 comprises less than 5% trisulfide variants, such as less than 4% trisulfide variants, such as less than 3% trisulfide variants, particularly less than 2% trisulfide variants, more particularly less than 1% trisulfide variants.

[0079] In one embodiment, provided herein is a method for producing an insulin-like growth factor 1 (IGF-1) protein complex, comprising the steps of providing a composition comprising recombinant IGF-1, adding an antioxidant to the composition, and purifying the IGF-1 from the composition.

[0080] In one embodiment, a method is provided herein comprising the step of adding an antioxidant, wherein the antioxidant is methionine.

[0081] In one embodiment, a method is provided herein comprising adding an antioxidant, wherein the antioxidant is sodium thiosulfate.

[0082] In one embodiment, provided herein is a method wherein the amount of oxidized IGF-1 species in a purified IGF-1 composition does not increase by more than 50%, such as not increasing by more than 45%, such as not increasing by more than 40%, particularly not increasing by more than 35%, and more particularly not increasing by more than 30% after 3 months of storage at a temperature of about 25°C.

[0083] In one embodiment, the amount of oxidized IGF-1 species in the purified IGF-1 composition is 25%; 20%; 15%; 10%; 5%; 3%; 2% after 3 months of storage at a temperature of about 25° C. *In one embodiment, a method is provided herein, wherein the amount of oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 1% after 3 months of storage at a temperature of about 25° C. In one embodiment, a method is provided herein, wherein the amount of oxidized IGF-1 species in the purified IGF-1 composition does not increase substantially after 3 months of storage at a temperature of about 25° C.

[0084] In some embodiments, the amount of oxidized IGF-1 species in the purified IGF-1 composition is, after 3 months of storage at a temperature of 2-8° C., 20%; 15%; 10%; 5%; 4%; 3%; 2% * Provided herein are methods in which the amount of oxidized IGF-1 species does not increase by more than 1% after 3 months of storage at a temperature of 2-8° C. In some embodiments, the amount of oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature of 2-8° C.

[0085] In some embodiments, the purified IGF-1 protein is * Provided herein are methods, in some embodiments, where the purified IGF-1 protein has less than 0.1% oxidized IGF-1 species.

[0086] In some embodiments, methods are provided herein where the recombinant IGF-1 protein is expressed in E. coli. In some embodiments, methods are provided herein where the recombinant IGF-1 protein is purified from inclusion bodies.

[0087] In some embodiments, the method further comprises adding an insulin-like growth factor binding protein (e.g., IGFBP-3) to the IGF-1 protein to form a protein complex. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in an equimolar amount, for example, in the range of 0.75 to 1.25:1 or 1:0.75-1.25. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 0.75 to 1.25 molar ratio. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 1:1 molar ratio. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 1.25-0.75 molar ratio.

[0088] In some embodiments, the composition comprises an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) produced by the method.

[0089] In one embodiment, the composition of the present disclosure comprises a surfactant, for example selected from polysorbate (e.g., polysorbate 80 or 20) or Triton-X, particularly at 0.01% or less, for example 0.008%, 0.0075%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003% or 0.

[0090] In one embodiment, the % of the formulation / composition is w / w or w / v or v / v.

[0091] In some embodiments, provided herein is a composition according to any embodiment of the disclosure comprising a protein complex comprising recombinant insulin-like growth factor 1 (rIGF-1) and recombinant insulin-like growth factor binding protein 3 (rIGFBP-3) in equimolar amounts, a polysorbate surfactant, e.g., 80 or 20, particularly polysorbate 20, at a concentration ranging from 0.0025 to 0.0075, e.g., about 0.005%, and a buffer comprising sodium acetate, acetic acid, and / or sodium chloride, wherein the composition has a pH of about 5.3-5.8, and the rIGF-1 / IGFBP-3 is at a concentration of 45-55 micrograms / mL, about 50 micrograms / mL, and less than 0.6% of the IGF-1 is present as an oxidized species, e.g., the composition comprises an antioxidant, e.g., methionine and / or sodium thiosulfate, among others, as described elsewhere herein.

[0092] In one embodiment, a lyophilized form of a composition according to any disclosure / embodiment described herein is provided, particularly including a bulking agent.

[0093] In one embodiment, provided herein is a lyophilized formulation comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein (e.g., IGFBP-3) in a 1:1 molar ratio, a bulking agent, a buffer, and a surfactant.

[0094] In one embodiment, the bulking agent is sucrose or trehalose. In one embodiment, the bulking agent is sucrose. In one formulation, the bulking agent is trehalose.

[0095] In one embodiment, the buffer is histidine or phosphate. In one embodiment, the buffer is histidine. In one embodiment, the buffer is phosphate. In one embodiment, the buffer is at pH 5.5-6.5.

[0096] In one embodiment, a formulation according to the present disclosure includes one or more amino acids, eg, independently selected from essential amino acids.

[0097] In one embodiment, there is provided the use of a composition according to the present disclosure in therapy.

[0098] In one embodiment there is provided a use of a composition according to the present disclosure in the manufacture of a medicament for the treatment, particularly for the treatment of infants, such as preterm infants, low gestational weight infants and / or post-pre-eclamptic infants, particularly for the treatment or prevention of BPD, ROP and / or IVH.

[0099] While the present invention has been described in conjunction with certain preferred embodiments thereof, it is to be understood that the foregoing description as well as the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0100] In one embodiment, the composition of the present disclosure is a liquid.

[0101] In one embodiment, the compositions of the present disclosure are stored at 2-8°C.

[0102] One embodiment includes reconstitution of a lyophilized formulation according to the present disclosure, for example, with an aqueous solution, including water for injection, saline or glucose, an isotonic solution.

[0103] In one embodiment, a formulation according to the present disclosure is isotonic prior to administration to a patient because, for example, it has been reconstituted with an aqueous solution (e.g., an isotonic solution) or because the final pharmaceutical product is isotonic.

[0104] In one embodiment, the composition according to the present disclosure does not contain benzalkonium chloride. This excipient is contraindicated for premature infants. In one embodiment, the formulation of the present disclosure does not contain a preservative.

[0105] In one embodiment, the formulations / compositions according to the present disclosure do not include material components that are toxic to infants, e.g., premature infants.

[0106] In one embodiment, stability refers to chemical stability, ie, stabilization against chemical degradation.

[0107] In one embodiment, stability refers to physical stability, for example, physical stability often results in aggregation, adsorption and / or changes in solubility (particularly increased insolubility).

[0108] In one embodiment, the compositions of the present disclosure minimize adsorption and / or loss of protein (ie, IGF-1 and / or IGFBP, such as IGFBP-3) during manufacture and / or administration.

[0109] In embodiments, stability refers to thermal stability, e.g., the ability to withstand an increase and / or decrease in temperature, e.g., the formulations of the present disclosure can withstand room temperature (e.g., about 25-27° C.) for at least 24 hours, and in particular can provide sufficient stability for administration, especially continuous administration.

[0110] In one embodiment, the compositions according to the present disclosure are resistant to degradation or changes caused by humidity.

[0111] In one embodiment, the composition according to the present disclosure is provided as a unit dose, for example a 24 hour dose, particularly for premature infants.

[0112] In one embodiment, the composition of the present disclosure, e.g., a liquid formulation, is provided in a disposable bag, in particular the formulation is suitable / ready for administration, which is advantageous because it minimizes the contact of the formulation with surfaces that may catalyze oxidation, and also minimizes contamination and infection to the patient.

[0113] In one embodiment, there is provided a concentrated liquid formulation according to the present disclosure provided in a vial, particularly a glass vial.

[0114] In one embodiment, a lyophilized formulation according to the present disclosure is provided in a vial, for example a glass vial.

[0115] In one embodiment, the contents of a vial or container (including a bag) filled with a formulation according to the present disclosure is under an inert gas, such as nitrogen.

[0116] In one embodiment, deamination pathways are minimized in protein complex formulations according to the present disclosure.

[0117] In one embodiment, the formulation of the present disclosure has a shelf life of at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months when stored under appropriate conditions.

[0118] In one embodiment, the formulations of the present disclosure are sterilized by filtration, for example, in which case loss of protein complexes is minimized during filtration with the formulations of the present disclosure.

[0119] In one embodiment, the pharmaceutical product has an outer foil packaging component to minimize ingress of moisture and / or exposure to light.

[0120] In one embodiment, a protein complex formulated (e.g., liquid formulation) and / or produced according to the present disclosure performs well in a stress test, e.g., a physical stress test, e.g., a stirring test, a shear test, an aggregation analysis, a liquid-air interface analysis, a dissolution test, and the like.

[0121] In one embodiment, there is provided a formulation and / or medicament obtainable from the methods disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] The present invention provides, inter alia, methods and compositions for reducing oxidation of formulations containing protein complexes including insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding proteins (e.g., IGFBP-3). Methods for reducing oxidative species (e.g., addition of antioxidants, lyophilization, disposable bags) provide improved formulations with greater potency and increased stability. Improved stability is achieved during storage at about 25° C. (e.g., about 23° C. to about 27° C.), or at about 40° C. (e.g., about 38° C. to about 42° C.), e.g., during storage for about 3-6 months. In some embodiments, the present invention provides improved IGF-1 / IGFBP-3 formulations that include antioxidants (e.g., methionine or sodium thiosulfate). In other embodiments, the present invention provides lyophilized formulations with reduced oxidative species. In other embodiments, high concentration proteins are formulated in vials for dilution prior to use.

[0123] In other embodiments, reduced accumulation of oxidative species and improved stability was achieved by the use of contact surfaces other than stainless steel, such as disposable bags, during compounding of the complex with the formulation solution. In summary, the present invention provides improved formulations of IGF-1 / IGFBP-3 that have the advantage, inter alia, of improved stability during storage, providing a safe and effective product for administration to neonates in the treatment of diseases of low birth weight infants.

[0124] In some aspects, provided herein is a composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, wherein the composition comprises an antioxidant, and the amount of oxidized IGF-1 species does not increase by more than 50% after 3 months of storage at a temperature of about 25° C.

[0125] In one embodiment, provided herein is a composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, wherein the amount of oxidized IGF-1 species does not increase by more than 20% after 3 months of storage at a temperature of 2-8° C.

[0126] The present invention provides, in some embodiments, compositions comprising methionine, e.g., at a concentration of 0.2 mM to 10 mM, as an antioxidant. In some embodiments, compositions are provided herein, in which the antioxidant is sodium thiosulfate, e.g., at a concentration of 0.2 mM to 10 mM. In some embodiments, compositions are provided herein, in which less than 5% of IGF-1 is present as oxidized species in the protein complex upon storage for 3 months. The amount of oxidized species is determined by reversed phase-ultra performance liquid chromatography (RP-UPLC) or by size exclusion chromatography-HPLC separation mode (SEC-HPLC). In some embodiments, compositions are provided herein, in which IGFBP-3 comprises less than 5% trisulfide variants.

[0127] In one embodiment, provided herein is a method for producing an insulin-like growth factor 1 (IGF-1) protein complex, comprising the steps of providing a composition comprising recombinant IGF-1, adding an antioxidant to the composition, and purifying the IGF-1 from the composition.

[0128] Provided herein is a method of producing an IGF-1 / IGFBP-3 complex comprising adding an antioxidant, wherein the antioxidant is methionine, wherein the antioxidant is added at a concentration of 0.2 mM to 10 mM. In one embodiment, provided herein is a method comprising adding an antioxidant, wherein the antioxidant is sodium thiosulfate, wherein the antioxidant is sodium thiosulfate, wherein the antioxidant is added at a concentration of 0.2 mM to 10 mM.

[0129] The present invention provides, in some embodiments, methods wherein the amount of oxidized IGF-1 species in a purified IGF-1 composition does not increase by more than 50% after 3 months of storage at a temperature of about 25° C. In some embodiments, methods are provided herein wherein the amount of oxidized IGF-1 species in a purified IGF-1 composition does not increase by more than 20% after 3 months of storage at a temperature of 2-8° C. In some embodiments, methods are provided herein wherein the purified IGF-1 protein has less than 5% oxidized IGF-1 species. In some embodiments, methods are provided herein wherein the recombinant IGF-1 protein is expressed in E. coli. In some embodiments, methods are provided herein wherein the recombinant IGF-1 protein is purified from inclusion bodies.

[0130] In some embodiments, the method further comprises adding insulin-like growth factor binding protein 3 (IGFBP-3) to the IGF-1 protein to form a protein complex. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in equimolar amounts, for example, in the range of 0.75 to 1.25:1 or 1:0.75-1.25. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 0.75 to 1.25 molar ratio. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 1:1 molar ratio. In some embodiments, the protein complex comprises IGF-1 and IGFBP-3 in a 1.25-0.75 molar ratio. In one embodiment, provided herein is a method for producing an insulin-like growth factor 1 (IGF-1) / IGFBP-3 protein complex, comprising the steps of providing a composition comprising recombinant IGF-1, recombinant IGFBP-3, adding an antioxidant to the composition, and purifying the IGF-1 / IGFBP-3.

[0131] In some embodiments, the composition comprises an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) produced by the method. In some aspects, provided herein is a composition comprising a protein complex comprising recombinant insulin-like growth factor 1 (rIGF-1) and recombinant insulin-like growth factor binding protein 3 (rIGFBP-3) in equimolar amounts, a polysorbate 20 surfactant at a concentration of about 0.005%, and a buffer comprising sodium acetate, acetic acid, and / or sodium chloride, wherein the composition has a pH of about 5.3-5.8, the rIGF-1 / IGFBP-3 is at a concentration of about 50 micrograms / mL, and less than 0.6% of the IGF-1 is present as an oxidized species.

[0132] In one embodiment, provided herein is a lyophilized formulation comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, a bulking agent, a buffer, and a surfactant.

[0133] In one embodiment, the bulking agent is sucrose or trehalose. In one embodiment, the bulking agent is sucrose. In one formulation, the bulking agent is trehalose.

[0134] In one embodiment, the buffer is histidine or phosphate. In one embodiment, the buffer is histidine. In one embodiment, the buffer is phosphate. In one embodiment, the buffer is at pH 5.5-6.5.

[0135] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section may apply to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0136] definition "Premature" or "preterm" or "low birth weight" or "preterm" or "premature infant", or grammatical equivalents, refers to the birth of an infant before 37 weeks of gestation or whose weight is 10% below average for the infant's gestational age. For example, an infant born between 22-37 weeks is considered premature.

[0137] In one embodiment, a premature infant (or newborn) as used herein refers to an infant born before, e.g., at 37 weeks or less (gestational age), e.g., 22-37 weeks, or 23-34 weeks or 24-29 weeks, particularly 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35 or 36 weeks gestational age. Generally, the patient, e.g., the infant, e.g., the premature infant, is a human.

[0138] The lowest weight infants (not necessarily the most preterm) are likely to benefit most from the treatments of the present disclosure.

[0139] Low gestational weight refers to infants in the lowest quartile of weight-for-gestational age.

[0140] Infants born after pre-eclampsia are self-explanatory.

[0141] "GA" or "gestational age" is a general term describing the progress of pregnancy, measured in weeks from the first day of a woman's last menstrual cycle to the current date.

[0142] In some embodiments, premature infants refer to infants born at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months prematurely. In some embodiments, premature infants refer to infants born at less than 32 weeks gestational age (GA). In some embodiments, premature infants refer to infants born at less than 28 weeks gestational age (GA). "PMA" or "postmenstrual age" refers to gestational age plus chronological age. "CA" or "corrected age" refers to chronological age reduced by the number of weeks born before 40 weeks gestation.

[0143] "IGF-I" refers to insulin-like growth factor I from any species, e.g., bovine, ovine, porcine, equine, and human, preferably human, and when referring to exogenous administration from any source, whether natural, synthetic, or recombinant, provided that it is bound to an IGF binding protein at the appropriate site. IGF-I can be produced recombinantly, e.g., as described in WO95 / 04076.

[0144] "IGFBP" or "IGF binding protein" refers to a protein or polypeptide from the insulin-like growth factor binding protein family that normally associates with, binds to, or complexes with IGF-I, whether circulating (i.e., in serum or tissues). Such binding proteins do not include receptors. Included in this definition are IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) or endothelial cell-specific molecule (ESM-1), as well as other proteins with high homology to IGFBPs. Mac25 is described, for example, in Swisshelm et al., Proc. Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol. Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al., Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalle et al., J. Biol. Chem., 271: 20458-20464 (1996).For other identified IGFBPs, see, e.g., EP 375,438 published June 27, 1990; EP 369,943 published May 23, 1990; WO 89 / 09268 published October 5, 1989; Wood et al., Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBO J., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411 (1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 published December 7, 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al., Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261: 8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235 (1988); WO89 / 08667 published September 21, 1989; WO89 / 09792 published October 19, 1989; and Binkert et al., EMBO J., 8: 2497-2502 (1989).

[0145] "IGFBP-3" refers to insulin-like growth factor binding protein 3. IGFBP-3 is a member of the insulin-like growth factor binding protein family. IGFBP-3 may be from any species, including bovine, ovine, porcine, and human, and may be in native sequence or mutant form, including but not limited to allelic variants of natural origin. IGFBP-3 may be from any source, whether natural, synthetic, or recombinant, provided that it binds to IGF-I at the appropriate site. IGFBP-3 may be produced recombinantly as described in PCT Publication WO 95 / 04076.

[0146] Formulations for IGF-1 and IGF binding proteins (e.g., IGFBP-3) are disclosed in WO2022 / 086953, which is incorporated herein by reference.

[0147] A "therapeutic composition" is defined herein to include IGF-I, its analogs, or its binding proteins, IGF-I in combination with IGFBP-3 (IGF-I / IGFBP-3 complex). Therapeutic compositions may also include other substances, such as water, minerals, carriers, such as proteins, and other excipients known to those skilled in the art. In some embodiments, the therapeutic composition includes a surfactant, such as polysorbate 20 (P20) or polysorbate 80 (P80).

[0148] An "analog" of IGF-I is a compound that has the same therapeutic effect as IGF-I in humans or animals. These can be either naturally occurring analogs of IGF-I (e.g., truncated IGF-I) or known synthetic analogs of IGF-I. For example, see U.S. Patent No. 5,473,054 for analog compounds of IGF-I.

[0149] An "antioxidant" is a compound included in the formulation that prevents or reduces the formation of oxidative species. In some embodiments, the antioxidant is selected from reducing agents, free radical scavengers, chelating agents, chain terminators, nitrogen, and the like. In some embodiments, the antioxidant is a reducing agent selected from the group consisting of L-methionine, sodium metabisulfite, sodium thiosulfate, and glutathione. In some embodiments, the antioxidant is a free radical scavenger selected from the group consisting of L-histidine, L-ascorbic acid, catalase, and platinum. In some embodiments, the antioxidant is a chelating agent selected from the group consisting of the sodium salt of EDTA dihydrate, diethylenetriaminepentaacetic acid (DTPA), and citric acid. In some embodiments, the antioxidant is a chain terminator consisting of L-methionine, L-cysteine, sodium thiosulfate, and BHT.

[0150] An "agonist" of IGF-I is a compound, including peptides, that can increase serum and tissue levels of IGF, especially IGF-I, in mammals, particularly humans. See, for example, US 6,251,865 for IGF agonist molecules.

[0151] "Lyophilization" refers to a process in which, after freezing and placing under vacuum, ice can change directly from a solid to a vapor without passing through a liquid phase, and water is removed from the product. "Lyophilized formulation" refers to a formulation that is lyophilized, for example, to improve stability during storage and increase shelf life, and is typically reconstituted prior to administration.

[0152] "Stability" refers to the degree to which a pharmaceutical product retains the characteristics and properties it had at the time of manufacture during its storage and use. Stability includes aspects related to its formulation, the stability of the IGF-1 and IGFBP (e.g., IGFBP3) material components, the (IGF-1 / IGFBP3) complex, the integrity and stability of the container and closure, manufacturing and processing conditions, packaging components, storage and shipping conditions, temperature, light, and humidity, as well as the expected duration and conditions of pharmacy shelf life and patient use. In some embodiments, stability refers to a reduction in the amount of undesirable species, such as oxidized species, a reduction in high molecular weight species that result in aggregation, and / or a reduction in low molecular weight species represented by degradation products. In some embodiments, stability refers to less than 10% undesirable species. In some embodiments, stability refers to less than 5% undesirable species. In some embodiments, stability refers to less than 2% undesirable species.

[0153] IGF-1 / IGFBP-3 IGF-1 or its agonists or analogs can be used to practice the present invention. IGF-I is a well-known regulator of postnatal growth and metabolism. See Baker J, Liu JP, Robertson EJ, Efstratiadis A. It has a molecular weight of approximately 7.5 kilodaltons (Kd). Most circulating IGFs bind to IGF-binding proteins, more specifically IGFBP-3. IGF-I can be measured in serum to diagnose abnormal growth-related conditions.

[0154] Typically, a therapeutic composition suitable for treating a disease described herein, including, for example, CLD, comprises IGF-1 and an IGF-1 binding protein, such as an IGF binding protein (IGFBP). At least six distinct IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. In some embodiments, a suitable therapeutic composition according to the present invention comprises IGF-1 and IGFBP-3. IGF-1 and IGFBP-3 may be used as a protein complex or separately. In some embodiments, IGF-1 and IGFBP (e.g., IGFBP-3) are complexed in equimolar amounts. In some embodiments, a therapeutic composition comprises mecasermin rinfavert. In some embodiments, a therapeutic composition comprises mecasermin rinfavert and a surfactant. In some embodiments, a therapeutic composition comprises mecasermin rinfavert, a surfactant, and an antioxidant. In some embodiments, a therapeutic composition comprises mecasermin rinfavert and polysorbate 20. In some embodiments, a therapeutic composition comprises mecasermin rinfavert and polysorbate 20 and an antioxidant. In some embodiments, the therapeutic composition comprises mecasermin rinfavert, polysorbate 20, and methionine. In some embodiments, the therapeutic composition comprises mecasermin rinfavert, polysorbate 20, and sodium thiosulfate. In some embodiments, the therapeutic composition comprises mecasermin rinfavert, polysorbate 80, and an antioxidant. In some embodiments, the therapeutic composition comprises mecasermin rinfavert, polysorbate 80, and methionine. In some embodiments, the therapeutic composition comprises mecasermin rinfavert, polysorbate 80, and sodium thiosulfate.

[0155] IGF-I and IGF-I binding proteins, such as IGFBP-3, can be purified from natural sources or produced by recombinant means.For example, the purification of IGF-I from human serum is well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad. Sci. USA 73:2365-2369). The production of IGF-I by recombinant process is shown in EP0128733 published in December 1984. IGFBP-3 can be purified from natural sources using a process such as that shown by Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261). Alternatively, IGFBP-3 can be synthesized recombinantly as discussed by Sommer et al., pp. 715-728, Modern Concepts Of Insulin-Like Growth Factors (E.M. Spencer, ed., Elsevier, NY, 1991). Recombinant IGFBP-3 binds to IGF-I in a 1:1 molar ratio (equimolar amounts).

[0156] Pharmaceutical Compositions and Therapeutic Uses In one embodiment, provided herein is a composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and an insulin-like growth factor binding protein (e.g., IGFBP-3), e.g., in a 1:1 molar ratio, wherein the composition comprises an antioxidant, and the amount of oxidized IGF-1 species does not increase by more than 50% after 3 months of storage at a temperature of about 25° C.

[0157] The formulations of the present invention are used in the treatment of patients suffering from diseases and complications of low birth weight infants, such as intraventricular hemorrhage (IVH), bronchopulmonary dysplasia (BPD) or chronic lung disease (CLD), such as, for example, CLD associated with low birth weight infants.For example, the present invention can be used to treat premature infants suffering from CLD or complications associated with CLD.In some embodiments, the present invention can be used to treat infants born at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months prematurely.In some embodiments, the present invention can be used to treat extremely premature infants.

[0158] In one embodiment of the invention, IGF-I is administered in combination with an IGF binding protein capable of binding to IGF-I and a surfactant. In some embodiments, the IGF binding protein capable of binding to IGF-I is IGF binding protein 3 (IGFBP-3). In some embodiments, the composition comprises a surfactant selected from polysorbate 20 or polysorbate 80. In some embodiments, the composition further comprises an antioxidant selected from methionine or sodium thiosulfate.

[0159] A composition comprising equimolar amounts of IGF-I and IGF-binding protein 3 may be used. In some embodiments, IGF-I and IGF-binding protein 3 are complexed prior to administration. The complex may be formed by mixing approximately equimolar amounts of IGF-I and IGF-binding protein 3 dissolved in a physiologically compatible carrier, such as normal saline or phosphate buffered saline. In some embodiments, a concentrated solution of recombinant human IGF-I and a concentrated solution of recombinant human IGF-binding protein 3 are mixed together for a sufficient time to form an equimolar complex. In some embodiments, recombinant human IGF-I and recombinant human IGF-binding protein 3 are combined during purification and complexed as described in WO96 / 40736.

[0160] Antioxidants Oxidation of protein residues arises from many different sources. Beyond the addition of specific antioxidants, prevention of oxidative protein damage involves careful control of many factors throughout the manufacturing process and storage of the product, such as atmospheric oxygen, temperature, light exposure, and chemical contamination. Therefore, the invention contemplates the use of pharmaceutical antioxidants, including but not limited to reducing agents, oxygen / free radical scavengers, or chelating agents.

[0161] Antioxidants are compounds included in formulations that prevent or reduce the formation of oxidizing species. Oxidation reactions transfer electrons from a substance to an oxidizing agent. During this process, some free radicals are generated, which initiate chain reactions that damage animal cells. Without wishing to be bound by any particular theory, it is contemplated that antioxidants slow down these chain reactions by scavenging free radical intermediates, and ultimately prevent other oxidation reactions by being oxidized themselves. Antioxidants often act as reducing agents, e.g., thiols or polyphenols.

[0162] Antioxidants in therapeutic protein formulations are, in one embodiment, water-soluble and remain active throughout the product shelf life. Reducing agents and oxygen / free radical scavengers act by scavenging reactive oxygen species in solution. Chelating agents such as EDTA are effective by binding trace metal contaminants that promote free radical formation.

[0163] In addition to the effectiveness of various excipients to prevent protein oxidation, there is concern that antioxidants themselves may cause other covalent or physical changes to proteins. For example, reducing agents can cause the disruption of intramolecular disulfide bonds, which can lead to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in many proteins and peptides (Akers MJ, and Defelippis M R. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors Pharmaceutical Science. Taylor and Francis, UK (1999));Fransson JR, J. Pharm. Sci. 86(9): 4046-1050 (1997); Yin J, et al., Pharm Res., 21(12): 2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light- and temperature-induced methionine-oxidation in rhuMab HER2; however, the formation of thiosulfate-protein adducts has also been reported (Lam XM, Yang JY, et al., J Pharm Sci. 86(11): 1250-5 (1997)). The selection of an appropriate antioxidant is made according to the specific stress and sensitivity of the protein. Antioxidants contemplated in certain embodiments include, but are not limited to, reducing agents and oxygen / free radical scavengers, EDTA, and sodium thiosulfate.

[0164] In some embodiments, the antioxidant is selected from reducing agents, free radical scavengers, chelating agents, chain terminators, nitrogen, etc. Methionine has been observed to be effective against many oxidative stresses (Lam XM, et al., J Pharm Sci., 86(11): 1250-5 (1997)).

[0165] In some embodiments, the antioxidant is a reducing agent selected from the group consisting of L-methionine, sodium metabisulfite, sodium thiosulfate, and glutathione. In some embodiments, the antioxidant is a free radical scavenger selected from the group consisting of L-histidine, L-ascorbic acid, catalase, and platinum. In some embodiments, the antioxidant is a chelating agent selected from the group consisting of the sodium salt of EDTA dihydrate, diethylenetriaminepentaacetic acid (DTPA), and citric acid. In some embodiments, the antioxidant is a chain terminator consisting of L-methionine, L-cysteine, sodium thiosulfate, and BHT. In some embodiments, oxidation is reduced by repeated vacuum application and addition or operation under nitrogen.

[0166] In some embodiments, the composition comprises 0.05 mM methionine, 0.10 mM methionine, 0.15 mM methionine, 0.20 mM methionine, 0.25 mM methionine, 0.30 mM methionine, 0.35 mM methionine, 0.40 mM methionine, 0.45 mM methionine, 0.50 mM methionine, 0.55 mM methionine, 0.60 mM methionine, 0.65 mM methionine, 0.70 mM methionine, 0.75 mM methionine, 0.80 mM methionine, 0.85 mM methionine, 0.90 mM methionine, 0.95 mM methionine, or 1 mM methionine.

[0167] In some embodiments, the composition comprises 1 mM methionine, 2 mM methionine, 3 mM methionine, 4 mM methionine, 5 mM methionine, 6 mM methionine, 7 mM methionine, 8 mM methionine, 9 mM methionine, 10 mM methionine, 11 mM methionine, 12 mM methionine, 13 mM methionine, 14 mM methionine, 15 mM methionine, 16 mM methionine, 17 mM methionine, 18 mM methionine, 19 mM methionine, or 20 mM methionine.

[0168] In some embodiments, the composition comprises 25 mM methionine, 30 mM methionine, 35 mM methionine, 40 mM methionine, 45 mM methionine, 50 mM methionine, 55 mM methionine, 60 mM methionine, 65 mM methionine, 70 mM methionine, 75 mM methionine, 80 mM methionine, 85 mM methionine, 90 mM methionine, 95 mM methionine, or 100 mM methionine.

[0169] In some embodiments, the composition comprises 0.05 mM sodium thiosulfate, 0.10 mM sodium thiosulfate, 0.15 mM sodium thiosulfate, 0.20 mM sodium thiosulfate, 0.25 mM sodium thiosulfate, 0.30 mM sodium thiosulfate, 0.35 mM sodium thiosulfate, 0.40 mM sodium thiosulfate, 0.45 mM sodium thiosulfate, 0.50 mM sodium thiosulfate, 0.55 mM sodium thiosulfate, 0.60 mM sodium thiosulfate, 0.65 mM sodium thiosulfate, 0.70 mM sodium thiosulfate, 0.75 mM sodium thiosulfate, 0.80 mM sodium thiosulfate, 0.85 mM sodium thiosulfate, 0.90 mM sodium thiosulfate, 0.95 mM sodium thiosulfate, or 1 mM sodium thiosulfate.

[0170] In some embodiments, the composition comprises 1 mM sodium thiosulfate, 2 mM sodium thiosulfate, 3 mM sodium thiosulfate, 4 mM sodium thiosulfate, 5 mM sodium thiosulfate, 6 mM sodium thiosulfate, 7 mM sodium thiosulfate, 8 mM sodium thiosulfate, 9 mM sodium thiosulfate, 10 mM sodium thiosulfate, 11 mM sodium thiosulfate, 12 mM sodium thiosulfate, 13 mM sodium thiosulfate, 14 mM sodium thiosulfate, 15 mM sodium thiosulfate, 16 mM sodium thiosulfate, 17 mM sodium thiosulfate, 18 mM sodium thiosulfate, 19 mM sodium thiosulfate, or 20 mM sodium thiosulfate.

[0171] In some embodiments, the composition comprises 25 mM sodium thiosulfate, 30 mM sodium thiosulfate, 35 mM sodium thiosulfate, 40 mM sodium thiosulfate, 45 mM sodium thiosulfate, 50 mM sodium thiosulfate, 55 mM sodium thiosulfate, 60 mM sodium thiosulfate, 65 mM sodium thiosulfate, 70 mM sodium thiosulfate, 75 mM sodium thiosulfate, 80 mM sodium thiosulfate, 85 mM sodium thiosulfate, 90 mM sodium thiosulfate, 95 mM sodium thiosulfate, or 100 mM sodium thiosulfate.

[0172] In some embodiments, the composition further comprises a buffer comprising sodium acetate, acetic acid, and / or sodium chloride. In some embodiments, the composition further comprises a buffer comprising sodium acetate or acetic acid. In some embodiments, the composition further comprises a buffer comprising sodium chloride.

[0173] In some embodiments, the sodium acetate or acetic acid is present at a concentration of about 10-100 mM. In some embodiments, the sodium acetate or acetic acid is present at a concentration of about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, the sodium acetate or acetic acid is present at a concentration of about 50 mM.

[0174] In some embodiments, sodium chloride is present at a concentration of about 20 mM to 200 mM. In some embodiments, sodium chloride is present at a concentration of about 20 mM, about 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM. In some embodiments, sodium chloride is present at a concentration of about 105 mM.

[0175] In some embodiments, the composition has a pH of about 5.0-6.0. In some embodiments, the pH is about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, or about 7.0. In some embodiments, the composition has a pH of about 5.1, about 5.3, about 5.5, about 5.7, about 5.9, about 6.1, about 6.3, about 6.5, about 6.7, or about 6.9. In some embodiments, the composition has a pH of about 5.5.

[0176] Freeze drying In one embodiment, the formulations containing IGF-1 / IGFBP (e.g., IGFBP-3) are lyophilized prior to administration. The lyophilization cycle, in one embodiment, consists of three steps: freezing, primary drying, and secondary drying. In the freezing step, the solution is cooled and ice formation is initiated. This step also induces the crystallization of the bulking agent. The ice is sublimated in the primary drying step, which is performed by reducing the chamber pressure below the vapor pressure of the ice, using a vacuum, and introducing heat to promote sublimation. Finally, the adsorbed or bound water is removed at elevated shelf temperatures under reduced chamber pressure in the secondary drying step. The process produces a material known as a lyophilized cake. The cake can then be reconstituted with either sterile water for injection or a suitable diluent.

[0177] The lyophilization cycle not only determines the final physical state of the excipient, but also influences other parameters such as reconstitution time, appearance, stability, and final water content. The composition structure in the frozen state progresses through several transitions (e.g., glass transition, wetting, and crystallization) that occur at specific temperatures, and the structure can be used to understand and optimize the lyophilization process. The glass transition temperature (Tg and / or Tg') can provide information about the physical state of the solute and can be determined by differential scanning calorimetry (DSC). Tg and Tg' are important parameters that must be considered when designing a lyophilization cycle. For example, Tg' is important for primary drying. Furthermore, in the dried state, the glass transition temperature provides information about the storage temperature of the final product. For example, in some embodiments, the lyophilization run is performed using a lyostar freeze dryer under a lyophilization cycle consisting of a primary drying temperature of -25°C and a secondary drying temperature of 25°C. In some embodiments, the lyophilization run is performed using a lyostar freeze dryer under a lyophilization cycle consisting of a primary drying temperature of -20°C and a secondary drying temperature of 25°C.

[0178] Formulations and excipients Excipients are additives that confer or enhance the stability and delivery of pharmaceuticals (e.g., IGF-1 / IGFBP (e.g., IGFBP-3) protein complexes). Excipients are essential components of the formulation and therefore must be safe and well tolerated by patients. For protein drugs, the selection of excipients is particularly important because they can affect both the efficacy and immunogenicity of the drug. Thus, protein formulations need to be developed with the appropriate selection of excipients that provide suitable stability, safety, and marketability.

[0179] In some embodiments, a pharmaceutical composition is provided comprising a composition described herein and one or more suitable pharmaceutical excipients.

[0180] In some embodiments, the composition comprises polysorbate 20. In some embodiments, the polysorbate 20 surfactant is present at a concentration of about 0.001% to 2.4% v / v. In some embodiments, the polysorbate 20 surfactant is present at a concentration of about 0.2% to 0.4% v / v. In some embodiments, the polysorbate 20 surfactant is present at a concentration of about 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.05%, 0.10%, 0.15%, 0.2%, 0.5%, 0.7%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, or 2.4% v / v. In some embodiments, the polysorbate 20 surfactant is present at a concentration of about 0.0025%, about 0.005%, or about 0.0075% v / v. In some embodiments, polysorbate 20 is present at a concentration of 0.005%.

[0181] In some embodiments, the composition comprises polysorbate 80. In some embodiments, the polysorbate 80 surfactant is present at a concentration of about 0.001% to 2.4% v / v. In some embodiments, the polysorbate 80 surfactant is present at a concentration of about 0.2% to 0.4% v / v. In some embodiments, the polysorbate 80 surfactant is present at a concentration of about 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.05%, 0.10%, 0.15%, 0.2%, 0.5%, 0.7%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, or 2.4% v / v. In some embodiments, the polysorbate 80 surfactant is present at a concentration of about 0.0025%, about 0.005%, or about 0.0075% v / v. In some embodiments, polysorbate 80 is present at a concentration of 0.005%.

[0182] The lyophilized formulation, in one embodiment, is composed of at least one or more of a buffer, a bulking agent, a stabilizer, and a surfactant. Appropriate buffering agents are included and the formulation is maintained within the pH stability zone during lyophilization.

[0183] The challenge in developing protein formulations for proteins is to stabilize the product against the stresses of manufacturing, shipping and storage. The role of formulation excipients is to provide stabilization against these stresses. Excipients are also used to reduce the viscosity of high concentration protein formulations to enable their delivery and enhance patient convenience. In general, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to mitigate the effects of specific stresses or to control the specific susceptibility of a particular protein. Other excipients have a more general effect on the physical and covalent stability of proteins. The excipients described herein are organized by either their chemical type or their functional role in the formulation. A brief description of the mode of stabilization is provided in discussing each excipient type. The amount and type of salts included in the biopharmaceuticals of the invention are selected based on the desired osmolality (i.e., isotonic, hypotonic or hypertonic) of the final solution as well as the amount and osmolality of other components included in the formulation.

[0184] As an example, the inclusion of about 5% sorbitol can achieve isotonicity, while about 9% sucrose excipient is required to achieve isotonicity. The selection of the amount or concentration range of one or more excipients that can be included in the biologics of the invention is exemplified above by reference to salts, polyols and sugars. However, those skilled in the art will understand that the considerations described herein and further exemplified by reference to specific excipients are equally applicable to all types and combinations of excipients, such as salts, amino acids, other isotonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, cryoprotectants, antioxidants, metal ions, chelating agents and / or preservatives.

[0185] Additionally, when a particular excipient is reported in molar concentration, one of skill in the art will recognize that the equivalent percent (%) w / v of the solution (e.g., (grams of substance in solution sample / mL of solution) x 100%) is also contemplated.

[0186] In some embodiments, the concentration of bulking agent can be reduced, e.g., where there is a high protein concentration, or, e.g., where there is a high stabilizer concentration, in some embodiments, the concentration of stabilizer will be adjusted accordingly (i.e., an "osmolality adjusting" amount of stabilizer will be used) to maintain the isotonicity of a particular formulation in the absence of bulking agent.

[0187] A comparison of contemplated excipient components for liquid and lyophilized protein formulations is provided in Table A. [Table 1]

[0188] Buffers and buffering agents It is usually observed that the stability of pharmacologically active protein formulations is maximum in a narrow pH range. This pH range of optimal stability needs to be identified early during preformulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this endeavor. Once the formulation is complete, the protein must be manufactured and maintained throughout its shelf life. Thus, buffers are most often used to control the pH in the formulation.

[0189] The buffering capacity of a buffer species is greatest at a pH equal to its pKa and decreases as the pH increases or decreases away from this value. Ninety percent of the buffering capacity is within one pH unit of its pKa. Buffering capacity also increases proportionally with increasing buffer concentration.

[0190] When selecting a buffer, the buffer type and its concentration need to be defined based on its pKa and the desired formulation pH. It is also important to ensure that the buffer is compatible with the protein and other formulation excipients and does not catalyze any degradation reactions. Another aspect to consider is the tingling and irritation that the buffer may induce upon administration. For example, citrate is known to cause a tingling sensation upon injection, especially when administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer period than when administered via IV routes, where the formulation is rapidly diluted in the blood upon administration. In formulations administered by direct IV infusion, the total amount of buffer (and any other formulation components) needs to be monitored, and potassium ions administered in the form of potassium phosphate buffer must be monitored for cardiovascular effects.

[0191] Buffers for lyophilized formulations require additional consideration. Some buffers, such as sodium phosphate, can crystallize from the protein amorphous phase during freezing, resulting in a pH shift. Other common buffers, such as acetate and imidazole, can sublime or evaporate during the lyophilization process, thus shifting the pH of the formulation during lyophilization or after reconstitution.

[0192] The buffer system present in the composition is physiologically compatible and is selected to maintain the desired pH of the pharmaceutical formulation. In one embodiment, the pH of the solution is pH 2.0 to pH 12.0. For example, the pH of the solution may be 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or 12.0.

[0193] The pH buffering compound may be present in any suitable amount to maintain the pH of the formulation at a predetermined level. In one embodiment, the pH buffering concentration is 0.1 mM to 500 mM (1M). For example, it is contemplated that the pH buffering agent is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM.

[0194] Exemplary pH buffering agents used to buffer the formulations set forth herein include, but are not limited to, organic acids, glycine, histidine, glutamate, succinate, phosphate, acetate, citrate, Tris, HEPES, and amino acids or mixtures of amino acids, such as, but not limited to, aspartic acid, histidine, and glycine. In one embodiment of the invention, the buffering agent is citrate.

[0195] Stabilizers and bulking agents In one embodiment, a stabilizer (or a combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or turbid solution upon reconstitution indicates that the protein has precipitated or at least aggregated. The term "stabilizer" refers to an excipient that can prevent aggregation or physical degradation, such as chemical degradation (e.g., autolysis, deamidation, oxidation, etc.) in aqueous conditions. Contemplated stabilizers include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, polyhydroxy compounds, such as polysaccharides, such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrollidene, cellulose and hyaluronic acid, sodium chloride. In the present formulations, the stabilizers are incorporated at a concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. In one embodiment, mannitol and trehalose are used as stabilizers.

[0196] In further embodiments, the formulation also includes an appropriate amount of bulking agent and osmolality adjuster.Bulking agents include, for example, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol.In one embodiment, the bulking agent is mannitol.The bulking agent is incorporated at a concentration of about 0.1, 0.5, 0.7, 0.80.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000mM.

[0197] Surfactants Proteins have a high tendency to interact with surfaces, making them susceptible to adsorption and denaturation at air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been observed to be inversely related to protein concentration and results in the formation of soluble and insoluble protein aggregates or loss of protein from solution due to adsorption to surfaces. In addition to container surface adsorption, surface-induced degradation is exacerbated by physical agitation, which may be experienced during product shipping and handling.

[0198] Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to outcompete proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies the interfacial positions (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentration (typically about the detergent's critical micelle concentration), the surface layer of surfactant molecules acts to prevent protein molecules from adsorbing at the interface. Thus, surface-induced degradation is minimized. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, namely polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that confers hydrophobicity to the molecule, C-12 and C-18, respectively. Thus, polysorbate 80 is more surface-active and has a lower critical micelle concentration than polysorbate 20.

[0199] Detergents affect the thermodynamic conformational stability of proteins. The hydrophobic tails of detergent molecules may be involved in specific binding to partially or fully unfolded protein states. These types of interactions would cause a shift in the conformational equilibrium towards more extended protein states (i.e., increased exposure of the hydrophobic parts of the protein molecule in the complement to the bound polysorbate). Alternatively, if the protein native state exhibits some hydrophobic surface, detergents binding to the native state could stabilize that conformation.

[0200] Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, especially methionine. The possibility of oxidative damage resulting from the addition of stabilizers emphasizes the point that the lowest effective concentration of excipients should be used in the formulation. For surfactants, the effective concentration for a given protein depends on the mechanism of stabilization.

[0201] Surfactants are also added in an appropriate amount to prevent surface-related aggregation phenomena during freezing and drying. Thus, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, such as surfactants derived from natural amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholic acid salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN®-20, and TWEEN®-80. Surfactants also include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, soybean lecithin and other phospholipids, such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC) and (dioleylphosphatidylglycerol) DOPG; sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Thus, compositions are further provided that contain these surfactants individually or as mixtures in different ratios. In one embodiment of the invention, the surfactant is TWEEN®-80. In the present formulation, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 g / L.In some embodiments of the provided formulations, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g / L. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is 0.005% polysorbate 20. In some embodiments, the surfactant is 0.005% polysorbate 80.

[0202] salt Salts are often added to increase the ionic strength of a formulation, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in various ways. Ions can stabilize the native state of a protein by binding to charged residues on the surface of the protein. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (-CONH-). Salts can also stabilize the native conformation of a protein by shielding repulsive electrostatic interactions between residues within the protein molecule. Salts in protein formulations can also shield attractive electrostatic interactions between protein molecules that can lead to protein aggregation and insolubility. In the provided formulations, the salt concentrations are 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300-500 mM.

[0203] Other Common Excipient Ingredients amino acid Amino acids find versatile use in protein formulations as buffers, bulking agents, stabilizers and antioxidants. Thus, in one embodiment, histidine and glutamic acid are used to buffer protein formulations at pH ranges of 5.5-6.5 and 4.0-5.5, respectively. The imidazole group of histidine has a pKa=6.0, and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, making these amino acids suitable for buffering in their respective pH ranges. Glutamic acid is particularly useful in such cases. Histidine is commonly found in commercial protein formulations, and this amino acid provides an alternative to citrate, a buffer known to be tingly upon injection. Interestingly, histidine also has a stabilizing effect on aggregation when used at high concentrations in both liquid and lyophilized presentations, reducing the viscosity of high protein concentration formulations.

[0204] In various aspects, formulations are provided that include one or more of the amino acids glycine, proline, serine, arginine, and alanine, which have been shown to stabilize proteins by a mechanism of preferential exclusion. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be an effective agent in preventing aggregation and has been used in both liquid and lyophilized formulations. In some embodiments, the amino acid concentration is 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300-500 mM.

[0205] Metal ions Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical decomposition reactions in proteins.However, certain metal ions are cofactors for proteins and are included in the formulation when present in the suspension formulation of proteins, where they form coordination compounds (e.g., zinc suspension of insulin).In addition, magnesium ions (10-120mM) are proposed to prevent the isomerization of aspartic acid to isoaspartic acid.

[0206] Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and Factor VIII. In the case of rhDNase, Ca+2 ions (up to 100 mM) increased the stability of the enzyme through specific binding sites. Indeed, removal of calcium ions from the solution using EGTA caused increased deamidation and aggregation. However, this effect was only observed with Ca+2 ions; other divalent cations Mg+2, Mn+2 and Zn+2 were observed to destabilize rhDNase. A similar effect was observed in Factor VIII. Ca+2 and Sr+2 ions stabilized the protein, whereas other ions such as Mg+2, Mn+2 and Zn+2, Cu+2 and Fe+2 destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997)). In another study with factor VIII, a significant increase in the rate of aggregation was observed in the presence of Al+3 ions (Derrick TS, et al., J. Pharm. Sci., 93(10): 2549-57 (2004)). Other excipients such as buffer salts are often contaminated with Al+3 ions, indicating the need to use excipients of appropriate quality in formulated products.

[0207] Preservatives Preservatives are required when developing multi-use parenteral formulations that require more than one extraction from the same container. Their primary function is to prevent microbial growth and ensure product sterility throughout the drug's shelf life or use. Commonly used preservatives include, but are not limited to, benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use, developing protein formulations containing preservatives can be difficult. Preservatives most often have a destabilizing effect on proteins (aggregation), which is the main factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm Sci., 94(2): 382-96 (2005)). To date, most protein drugs have been formulated solely for single-use. When multi-dose formulations are possible, they have the added advantage of allowing for patient convenience and increased marketability. In the case of human growth hormone (hGH), development of preserved formulations has led to the commercialization of more convenient, multi-use injection pens; for example, Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (EliLilly) is formulated with m-cresol.

[0208] Several aspects need to be considered during formulation development of a preserved dosage form. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in the dosage form at a range of concentrations that impart antimicrobial efficacy without compromising protein stability. For example, three preservatives were successfully screened in the development of a liquid formulation for Interleukin-1 Receptor (Type I) using Differential Scanning Calorimetry (DSC). The preservatives were ranked based on their impact on stability at concentrations commonly used in commercial products.

[0209] Developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This significantly reduces the time the preservative is in contact with the protein, minimizing the associated stability risks. In liquid formulations, preservative effectiveness and stability must be maintained over the entire product shelf life (approximately 18-24 months). An important point to note is that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0210] Some preservatives may cause injection site reactions, which is another factor that needs to be considered when selecting a preservative. In a clinical trial focused on the evaluation of preservatives and buffers in Norditropin, it was observed that pain sensation was lower in formulations containing phenol and benzyl alcohol compared to formulations containing m-cresol (Kappelgaard AM, Horm Res. 62 Suppl 3:98-103 (2004)). Interestingly, among the commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun D A., Anesth Analg., 100(3): 683-6 (2005)). In various embodiments, the use of a preservative provides benefits that outweigh any side effects.

[0211] Method of preparation The present invention further contemplates a method for preparing a pharmaceutical formulation, the method further comprising one or more of the following steps: adding a stabilizing agent as described herein to the mixture prior to lyophilization, adding at least one agent selected from a bulking agent, an osmolality adjusting agent, and a surfactant, each of which is described herein, to the mixture prior to lyophilization.

[0212] Standard reconstitution practice for lyophilized material is to add a volume of purified water or sterile water for injection (WFI) (typically equal to the volume removed during lyophilization), although dilute solutions of antimicrobial agents are sometimes used in the production of drug products for parenteral administration [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)].

[0213] The lyophilized material can be reconstituted as an aqueous solution with a variety of aqueous carriers, such as sterile water for injection, water with a preservative for multiple dose use, or water with an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound mixed with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, such excipients are suspending agents, such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing agents or wetting agents are natural phosphatides, such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as, but not limited to, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as, but not limited to, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, the aqueous suspension also contains one or more preservatives, such as, but not limited to, ethyl, or n-propyl, p-hydroxybenzoates.

[0214] The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.The formulations can conveniently be provided in unit dosage form, for example, single-use bags containing a single dose (e.g., a dose for 24 hours), tablets and sustained release capsules, and liposomes, and can be prepared by any method well known in the art of pharmacy.See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa. (17th Edition, 1985).In some embodiments, the composition is administered intravenously.

[0215] Such preparative methods include the step of bringing into association the molecules to be administered, such as the carriers which constitute one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0216] Compositions of the present invention suitable for oral administration can be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion, or loaded into liposomes, and as a bolus, etc.

[0217] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Preparations may be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0218] In some embodiments, the methods disclosed herein provide for parenteral administration of IGF-I complexed with an IGF binding protein (e.g., IGFBP-3) in the presence of a surfactant, e.g., 0.005% polysorbate 20 or polysorbate 80, and an antioxidant, e.g., sodium thiosulfate and methionine, to an infant in need of such treatment. Parenteral administration includes, but is not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraperitoneal (IP), intranasal, and inhalation routes. In some embodiments, the IGF-I / IGFBP-3 composition is administered intravenously. IV, IM, SC, and IP administration may be by bolus or infusion, and may also be by slow release implantation devices, including, but not limited to, pumps, slow release formulations, and mechanical devices. The formulation, route and method of administration, and dosage depend on the disorder being treated and the patient's medical history. Thus, in some embodiments, the methods disclosed herein provide for intravenous administration to an infant in need of such treatment of IGF-I complexed to an IGF binding protein (e.g., IGFBP-3) in the presence of a surfactant, e.g., 0.005% polysorbate 20 or polysorbate 80. In some embodiments, the methods disclosed herein provide for subcutaneous administration to an infant in need of such treatment of IGF-I complexed to an IGF binding protein 3 in the presence of a surfactant, e.g., 0.005% polysorbate 20 or polysorbate 80, and an antioxidant, e.g., methionine or sodium thiosulfate.

[0219] In some embodiments, the composition comprising rIGF-1 / IGFBP-3 is present at a concentration of about 10 micrograms / mL-100 micrograms / mL. For example, in some embodiments, the composition comprising rIGF-1 / IGFBP-3 is present at a concentration of about 45-55 micrograms / mL. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 is at a concentration of about 50 micrograms / mL. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 10-100 micrograms / mL is suitable for intravenous administration. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 45-55 micrograms / mL is suitable for intravenous administration. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 50 micrograms / mL is suitable for intravenous administration.

[0220] In some embodiments, the composition comprising rIGF-1 / IGFBP-3 is present at a concentration of about 1000 micrograms / mL-5000 micrograms / mL. For example, in some embodiments, the composition comprising rIGF-1 / IGFBP-3 is present at a concentration of 2000 micrograms / mL-3000 micrograms / mL. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 is present at a concentration of about 2500 micrograms / mL. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 1000-5000 micrograms / mL is suitable for intravenous administration. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 2000 micrograms / mL-3000 micrograms / mL is suitable for intravenous administration. In some embodiments, the composition comprising rIGF-1 / IGFBP-3 at a concentration of about 2500 micrograms / mL is suitable for intravenous administration.

[0221] The pharmaceutical composition according to the present invention may be administered in various doses. For example, a suitable dosage may be in the range of about 100 to 1000 micrograms / kg / 24 hours. In some embodiments, a suitable dosage may be about 100 micrograms / kg / 24 hours, 150 micrograms / kg / 24 hours, 200 micrograms / kg / 24 hours, 250 micrograms / kg / 24 hours, 300 micrograms / kg / 24 hours, 350 micrograms / kg / 24 hours, 400 micrograms / kg / 24 hours, 450 micrograms / kg / 24 hours, 500 micrograms / kg / 24 hours, 550 micrograms / kg / 24 hours, 600 micrograms / kg / 24 hours, 650 micrograms / kg / 24 hours, 700 micrograms / kg / 24 hours, 750 micrograms / kg / 24 hours, 800 micrograms / kg / 24 hours, 850 micrograms / kg / 24 hours, 900 micrograms / kg / 24 hours, 950 micrograms / kg / 24 hours, or 1000 micrograms / kg / 24 hours or more. In some embodiments, a suitable dosage is about 250 micrograms / kg / 24 hours. In some embodiments, a suitable dosage is about 400 micrograms / kg / 24 hours. In some embodiments, a suitable dosage is about 750 micrograms / kg / 24 hours. In some embodiments, a suitable dosage is about 1000 micrograms / kg / 24 hours. In some embodiments, a pharmaceutical composition according to the present invention is administered from birth up to about 23-34 weeks postmenstrual age (PMA), up to about 28-32 weeks PMA, up to about 29 weeks+6 days PMA.

[0222] For parenteral or oral administration, the composition of the complex may be a semi-solid or liquid preparation, e.g., a liquid, a suspension, etc. A physiologically compatible carrier is one that is non-toxic to a recipient at the dosage and concentration used, and is compatible with the other ingredients of the formulation. For example, the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to polypeptides. Thus, physiologically compatible carriers include, but are not limited to, normal saline, serum albumin, 5% dextrose, plasma preparations, and other protein-containing solutions. Optionally, the carrier may also contain a detergent or surfactant.

[0223] In some embodiments, a dry lyophilized powder is provided herein. In some embodiments, the composition can be reconstituted in sterile pharmaceutical grade water, saline or buffered water.

[0224] In some embodiments, a kit is provided for carrying out the methods described herein.In certain embodiments, the kit comprises a composition comprising rIGF-1, rIGFBP-3, a polysorbate surfactant, such as polysorbate 20 or polysorbate 80, and an antioxidant, such as methionine or sodium thiosulfate.In some embodiments, the kit comprises the composition described herein in a vial of glass or other suitable material.

[0225] In some embodiments, an article of manufacture is provided that includes packaging material and a pharmaceutical product contained within the packaging material. The packaging material includes a label indicating that the pharmaceutical product may be administered at an effective dose for a sufficient period of time to treat and / or prevent complications associated with preterm birth. The pharmaceutical product includes IGF-I, an agonist or an analog thereof, together with a pharma- ceutical acceptable carrier.

[0226] The values ​​and / or features in the example(s) may be used as the basis for an amendment to the claims, and the values ​​and / or features may be adopted without regard to other elements used in the example, i.e., the values ​​may be used alone.

[0227] Embodiments herein referring to "compositions" apply equally to other types of disclosures herein, such as methods and uses, and vice versa, unless otherwise indicated.

[0228] As employed herein, "is" means including.

[0229] In the context of this specification "including" should be interpreted as "including".

[0230] Embodiments of the invention comprising certain features / elements are also intended to extend to other embodiments that "consist of" or "consist essentially of" the associated element / feature.

[0231] Where technically appropriate, embodiments of the invention may be combined.

[0232] Technical references, such as patents and applications, are incorporated herein by reference.

[0233] Any embodiment specifically and explicitly described herein may be used alone or in combination with one or more additional embodiments to form a basis for disclaimer.

[0234] The background section contains technically relevant details that may be used as a basis for amendments.

[0235] The individual numerical values ​​in the examples generally relate to aspects and embodiments of the invention and therefore may be used as the basis for amendments to the claims.

[0236] The invention is further characterized by the following examples which are intended to be illustrative of the invention.

[0237] The drawings are for illustration only and are not intended to be limiting. [Brief description of the drawings]

[0238] [Figure 1] FIG. 1 is a graph showing the amount of oxidized rhIGF-1, expressed as a percentage area, as measured using reverse phase high performance liquid chromatography (RP-HPLC). [Diagram 2] 1 is a graph showing the amount of rhIGFBP-3, expressed as percentage area, as determined using RP-HPLC. [Diagram 3] FIG. 1 is a graph showing the amount of rhIGF-1 expressed as percentage area, as determined using RP-HPLC. [Figure 4A] FIG. 1 is a graph showing the change in oxidized rhIGF-1 in a formulation of 0.15 mg / mL protein by SEC-HPLC assessment after 3 months of storage in the presence of no antioxidant, either 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 4B] FIG. 1 is a graph showing the change in oxidized rhIGF-1 in formulations of 0.05 mg / mL protein by SEC-HPLC assessment after 3 months of storage in the presence of either no antioxidant, 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 5A] FIG. 1 is a graph showing the change in the amount of rhIGF-1 in a formulation of 0.15 mg / mL protein by SEC-HPLC assessment after 3 months of storage in the presence of no antioxidant, either 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 5B]FIG. 1 is a graph showing the change in the amount of rhIGF-1 in formulations of 0.05 mg / mL protein by SEC-HPLC assessment after 3 months of storage in the presence of either no antioxidant, 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 6A] FIG. 1 is a graph showing the change in the amount of rhIGF-BP3 in a formulation of 0.15 mg / mL protein by SEC-HPLC evaluation after 3 months of storage in the presence of no antioxidant, either 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 6B] FIG. 1 is a graph showing the change in the amount of rhIGF-BP3 in a formulation of 0.05 mg / mL protein by SEC-HPLC evaluation after 3 months of storage in the presence of either no antioxidant, 0.5 mM or 5 mM methionine, or 0.5 mM or 5 mM sodium thiosulfate. [Figure 7A] FIG. 1 is an SDS-PAGE gel showing the stability of IGF-1 / IGFBP-3 protein at 150 μg / mL after 3 months of storage at 2-8° C. in the absence of antioxidants and in the presence of methionine or sodium thiosulfate. [Figure 7B] FIG. 1 is an SDS-PAGE gel showing the stability of IGF-1 / IGFBP-3 protein at 50 μg / mL after 3 months of storage at 2-8° C. in the absence of antioxidants and in the presence of methionine or sodium thiosulfate. [Figure 7C] FIG. 1 is an SDS-PAGE gel showing the stability of IGF-1 / IGFBP-3 protein at 150 μg / mL after 3 months of storage at 2-8° C. in the absence of antioxidants and in the presence of methionine or sodium thiosulfate. [Figure 7D] FIG. 1 is an SDS-PAGE gel showing the stability of IGF-1 / IGFBP-3 protein at 50 μg / mL after 3 months of storage at 2-8° C. in the absence of antioxidants and in the presence of methionine or sodium thiosulfate. [Figure 8A] 1 shows the temperature profile of a freeze-drying run that has reached a predetermined set point. [Figure 8B]Figure 1 shows the pressure profile during the freeze-drying run. The Pirani pressure profile showed that sublimation was terminated after 34 hours of primary drying when the Pirani pressure reached a set point of 100 mTorr as measured by a capacitance manometer. Secondary drying led to further desorption of water and the Pirani pressure reached the set point again during secondary drying. [Figure 9A] 1 shows the baseline condition of drug product vials from a lyophilization run. [Figure 9B] Vials are shown after 2 weeks of storage at 60°C. [Figure 9C] Vials are shown after 4 weeks of storage at 60°C. [Figure 10A] Quantity of rhIGF-1 / rhIGFBP-3 protein complex by SE-UPLC at baseline, after 2 weeks and 4 weeks of storage compared to the liquid formulation is shown. [Figure 10B] The amount of high molecular weight species measured by SE-UPLC at baseline, after 2 weeks and after 4 weeks of storage compared to the liquid formulation is shown. [Figure 11A] The amount of rhIGFBP-3 measured by SE-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation is shown. [Figure 11B] The amount of rIGF-1 measured by SE-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation is shown. [Figure 11C] The amount of oxidized rIGF-1 measured by SE-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation is shown. [Figure 12A] A representative lyophilization run at baseline is shown. [Figure 12B] A representative formulation is shown after 4 weeks of storage at 60°C. [Figure 12C] A representative formulation is shown after 4 weeks of storage at 60°C. [Figure 13A] A representative lyophilization run at baseline is shown. [Figure 13B] A representative formulation is shown after 4 weeks of storage at 60°C. [Figure 13C] A representative formulation is shown after 4 weeks of storage at 60°C. [Figure 13D] A representative formulation is shown after 4 weeks of storage at 60°C. [Figure 14A] An SDS PAGE gel of a representative lyophilized formulation at baseline is shown. [Figure 14B] An SDS PAGE gel of a representative lyophilized formulation after 4 weeks of storage at 40° C. is shown. [Figure 15A] 1 shows the temperature profile of an exemplary freeze-drying run. [Figure 15B] 1 shows the pressure profile of an exemplary freeze-drying run. [Figure 16A] 1 shows a graph of percent rhIGF-1 / rhIGFBP-3 protein complex amount from an exemplary lyophilization run. [Figure 16B] 1 shows a graph of the amount of high molecular weight species from an exemplary lyophilization run. [Figure 17A] 1 shows a graph of the amount of rhIGFBP-3 measured by RP-UPLC at baseline, after 2 weeks of storage, and after 4 weeks of storage compared to the liquid formulation. [Figure 17B] A graph of the amount of rhIGF-1 measured by RP-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation is shown. [Figure 17C] A graph of the amount of oxidized rhIGF-1 measured by RP-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation is shown. [Figure 18A] 1 shows a graph of a representative lyophilized formulation at baseline. [Figure 18B] Graph of a representative formulation after 4 weeks storage at 40° C. is shown. [Figure 18C] Graph of a representative formulation after 4 weeks storage at 40° C. is shown. [Figure 18D] Graph of a representative formulation after 4 weeks storage at 40° C. is shown. [Figure 18E] Graph of a representative formulation after 4 weeks storage at 40° C. is shown. [Figure 18F] 1 shows a graph of a representative formulation after 48 hours of liquid storage at room temperature. [Figure 19A] Graphs of representative formulations at baseline are shown. [Figure 19B] Graph of a representative formulation after 4 weeks storage at 40° C. is shown. [Figure 19C] 1 shows a graph of a representative formulation after 48 hours of liquid storage at room temperature. [Figure 20A] 1 shows an SDS PAGE gel of a representative lyophilized protein formulation at baseline. [Figure 20B] 1 shows an SDS PAGE gel of a representative lyophilized protein formulation after 4 weeks of storage at 40° C.

[0239] Working Example While certain compounds, compositions and methods described herein have been specifically described according to certain embodiments, the following examples are only intended to illustrate the compounds of the invention and are not intended to be limiting thereof.

[0240] Example 1 Measurement of oxidized rIGF-1 by RP-UPLC after addition of antioxidants to IGF-1 / IGFBP-3 liquid formulations This example shows the amount of oxidized species in formulations containing insulin-like growth factor-1 / insulin-like growth factor binding protein-3 (rhIGF-1 / rhIGFBP-3) complex with or without added antioxidants.

[0241] In this example, L-methionine and sodium thiosulfate are used as exemplary antioxidants at concentrations of 5 mM and 50 mM. Mecasermin rinfavert (IGF-1 / rhIGFBP-3) is used at protein concentrations of 0.15 mg / mL and 0.50 mg / mL. Stability was measured after 3 months of storage at 2-8° C. and 25° C. Percent oxidized species (ox rhIGF-1, FIG. 1), rhIGF-1 (FIG. 2) and rhIGFBP-3 (FIG. 3) were measured by RP-UPLC after 3 months at 2-8° C. (e.g., 5° C.) and 25° C. [Table 2]

[0242] Results showed that at higher protein concentrations (0.15 mg / mL) protein was less susceptible to oxidative degradation, and that both methionine and sodium thiosulfate were effective at reducing the increase in oxidized rhIGF-1 species at all concentrations. At lower protein concentrations, L-methionine was more effective as an antioxidant. [Table 3] [Table 4]

[0243] Example 2 Measurement of oxidized rIGF-1 by SEC-HPLC after addition of antioxidants to IGF-1 / IGFBP-3 formulations This example shows the amount of oxidized species in formulations containing insulin-like growth factor-1 / insulin-like growth factor binding protein-3 (rhIGF-1 / rhIGFBP-3) complex with or without added antioxidants.

[0244] In this example, L-methionine and sodium thiosulfate are used as exemplary antioxidants at 5 mM and 50 mM concentrations. Mecasermin rinfavert (IGF-1 / rhIGFBP-3) is used at protein concentrations of 0.15 mg / mL and 0.50 mg / mL. Stability is measured after 3 months of storage at 2-8°C and 25°C. Percent oxidized species (ox rhIGF-1), rhIGF-1 and rhIGFBP-3 are measured by SEC-HPLC at baseline and after 3 months at 2-8°C (e.g., 5°C) and 25°C. Changes in oxidized rhIGF-1 by SEC assessment at baseline and after 3 months are shown at protein concentrations of 0.15 mg / mL (Figure 4A) and 0.05 mg / mL (Figure 4B). Changes in rhIGF-1 by SEC assessment at baseline and after 3 months are shown at protein concentrations of 0.15 mg / mL (Figure 5A) and 0.05 mg / mL (Figure 5B). Changes in oxidized rhIGFBP-3 by SEC assessment at baseline and 3 months are shown at protein concentrations of 0.15 mg / mL (FIG. 6A) and 0.05 mg / mL (FIG. 6B). [Table 5]

[0245] Results showed that the formulation was less susceptible to degradation at higher protein concentrations of 0.15 mg / mL compared to 0.05 mg / mL. Results also showed that both methionine and sodium thiosulfate were effective at all concentrations.

[0246] Furthermore, by SEC analysis, the results indicated that at lower protein concentrations, sodium thiosulfate was somewhat more effective as an antioxidant compared to methionine. [Table 6] [Table 7]

[0247] The data showed that both L-methionine and sodium thiosulfate can act as antioxidants.

[0248] No significant protein degradation was observed by SDS-PAGE at 2-8°C at 150 μg (Figure 7A) and 50 μg protein concentrations (Figure 7B), or at 25°C at 150 μg (Figure 7C) and 50 μg (Figure 7D) after 3 months.

[0249] Example 3. Comparison of oxidized rIGF-1 after addition of antioxidants to IGF-1 / IGFBP-3 formulations and storage at <-65°C and 2-8°C for 3 months This example measures the amount of oxidized rIGF-1 after the addition of antioxidants to an IGF-1 / IGFBP-3 formulation and after 3 months of storage at below -65°C and at 2-8°C. [Table 8]

[0250] The data in this example show that storage below -65°C (frozen) is preferred over storage at 2-8°C. [Table 9]

[0251] Example 4. Lyophilization of Mecasermin Linfavert (IGF-1 / IGFBP-3) This example shows the effect of lyophilization on the percent oxidation and stability of IGF-1 / IGFBP-3.

[0252] In this example, mecasermin linfavert pharmaceutical formulations using various buffer excipients and bulking agents were lyophilized and evaluated for physical properties and degradation.

[0253] In this example, the protein concentration of the IGF-1 / IGFBP-3 complex was 0.15 mg / mL (150 μg / ml) and polysorbate was added at 0.015%. Three formulation buffers, including histidine chloride, sodium phosphate and sodium citrate buffers, and two bulking agents, sucrose and trehalose, were compared (Table 9).

[0254] The results showed that the histidine buffer resulted in the least amount of high molecular weight (HMW) species formation. [Table 10]

[0255] Freeze-drying was performed under a freeze-drying cycle consisting of a primary drying temperature of -25°C and a secondary drying temperature of 25°C (Table 10). The running temperature profile is shown in Figure 8A and the pressure profile is shown in Figure 8B. [Table 11]

[0256] The lyophilization run yielded a homogenous white cake for all vials (Table 11, Figure 9A, Figure 9B, and Figure 9C). The lyophilized vials were reconstituted with 6.2 mL of normal saline and product quality was assessed by protein concentration using SoloVPE, pH, SE-UPLC, RP-UPLC, and reduced SDS-PAGE. Cake appearance and reconstitution time were also evaluated for the lyophilized formulations in addition to osmolality which was assessed at baseline for the formulated liquid drug product. [Table 12]

[0257] As can be seen from the results in Table 11, after two weeks of storage at 60° C., the cake containing sucrose melted, while the cake containing trehalose remained unchanged. [Table 13]

[0258] Cakes containing sucrose required increased reconstitution times, whereas reconstitution times were virtually unchanged for cakes containing trehalose (Table 12).

[0259] SE-UPLC separates the main peak from the high molecular weight aggregate peak (Figure 10A). Analysis by SE-UPLC showed that the use of trehalose as a bulking agent resulted in better protein stability than the use of sucrose. The HMW peak percentage for the sucrose formulations after 4 weeks of storage at 60°C ranged from 7.9% for formulation #3 to 20.2% for formulation #5 (Figure 10B). The increased aggregation indicates that trehalose is a better cryoprotectant than sucrose or that, as a result, lyophilized cakes containing sucrose, for example, collapse due to residual moisture in the cake. Comparison of the results for sodium citrate, histidine chloride, and sodium phosphate buffers showed that the drug product formulated in the histidine buffer resulted in the least amount of HMW species formation.

[0260] Freeze drying of mecasermin rinfavert formulations using either sucrose or trehalose as bulking agents resulted in a white, uniform cake. Some cake shrinkage was observed (approximately 10%-20% visual shrinkage for trehalose-containing formulations and approximately 10%-50% for sucrose-containing formulations). Heat stress storage at 60°C for 4 weeks was used to evaluate degradation of the lyophilized drug products. Degradation was least for the formulations containing trehalose.

[0261] The data from this example showed that trehalose performed better as a bulking agent than sucrose for the lyophilized presentation, and that histidine and phosphate buffers were preferred buffers compared to citrate buffer. The data also showed that the liquid form of the formulation before lyophilization was stable under refrigerated conditions for 48 hours.

[0262] Example 5. Lyophilization of Mecasermin Linfavert (IGF-1 / IGFBP-3) This example demonstrates the effect of pH on lyophilization of IGF-1 / IGFBP-3 in histidine buffer.

[0263] Mecasermin rinfavert (IGF-1 / IGFBP-3) was compared at three pH values, 5.5, 6.0, and 6.5, in histidine buffer (Table 13). Protein concentration before lyophilization was 0.15 mg / mL, and polysorbate was added at 0.015%. Trehalose was used as a bulking agent. The concentration of trehalose was reduced to 4% to reduce overall osmolality. Formulation #4 in Table 5 contained 1 mM methionine to evaluate the effect of methionine on oxidation levels. [Table 14]

[0264] The freeze-drying runs were carried out using a lyostar freeze-dryer under a freeze-drying cycle consisting of a primary drying temperature of -20°C and a secondary drying temperature of 25°C (Table 14). [Table 15]

[0265] Lyophilized drug product vials were subjected to heat stress conditions at 40°C for 2 and 4 weeks and 60°C for 2 weeks. One vial of each formulation was kept at room temperature for 48 hours as a liquid control without lyophilization. The lyophilization run yielded a homogenous firm white cake with shrinkage as assessed visually (20% at baseline, 20-30% after heat stress storage). Lyophilized vials were reconstituted with 6.2 mL of normal saline. Reconstitution times were less than 20 seconds at baseline for all formulations.

[0266] SE-UPLC analysis showed that the % peak area of ​​the complex of rhIGF-1 and rhIGFBP-3 was highest at pH values ​​6.0 and 6.5, and the high molecular weight species was lowest after 4 weeks of thermal storage at pH 6.0 followed by pH 6.5 (Figure 11A). The addition of methionine showed a small amount of high molecular weight species for liquid samples kept at room temperature for 48 hours (Figure 11A).

[0267] A representative lyophilization run at baseline is shown in Figure 12A. Two representative formulations after 4 weeks of storage at 60°C (Figures 12B and 12C). A representative lyophilization run at baseline in Figures 13A, 13B, and 13C shows a representative formulation after 4 weeks of storage at 60°C.

[0268] An SDS PAGE gel of a representative lyophilized formulation at baseline is shown in Figure 14A and compared to an SDS PAGE gel of a representative lyophilized formulation after 4 weeks of storage at 40°C (Figure 14B). Figure 15A shows the temperature profile of an exemplary lyophilization run. Figure 15B shows the pressure profile of an exemplary lyophilization run. Figure 16A shows a graph of the amount of percent rhIGF-1 / rhIGFBP-3 protein complex from an exemplary lyophilization run. Figure 16B shows a graph of the amount of high molecular weight species from an exemplary lyophilization run.

[0269] RP-UPLC data for all four formulations containing protein were similar (Figure 17A). Reduced oxidation was observed in samples containing 1 mM methionine (Figure 17A). Figure 17B shows a graph of the amount of rhIGF-1 measured by RP-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation. Figure 17C shows a graph of the amount of oxidized rhIGF-1 measured by RP-UPLC at baseline, after 2 weeks storage, and after 4 weeks storage compared to the liquid formulation. No significant differences were observed between pH 5.5, 6.0, and 6.5. Figure 18A shows a graph of a representative lyophilized formulation at baseline. Graphs of representative formulations after 4 weeks storage at 40°C are shown in Figures 18B, 18C, 18D, and 18E. Figure 18F shows a graph of a representative formulation after 48 hours of liquid storage at room temperature.

[0270] A graph of a representative formulation at baseline is shown in Figure 19A and compared to a graph of a representative formulation after 4 weeks of storage at 40°C (Figure 19B). Figure 19C shows a graph of a representative formulation after 48 hours of liquid storage at room temperature. Analysis by SDS-PAGE (Coomassie) showed no degradation (Figures 20A and 20B). Measured pH values ​​were within ±0.1 units of the expected values ​​after stressed storage (Table 12).

[0271] Overall, the data in this example showed that a 5.5-6.5 pH range was suitable for the formulation and that the addition of a small amount of methionine (1 mM) reduced the tendency for oxidation for the lyophilized representation and liquid control.

[0272] While certain compounds, compositions and methods described herein have been specifically described according to certain embodiments, the following examples are only intended to illustrate the compounds of the invention and are not intended to be limiting thereof.

[0273] The article "a" or "an" as used herein should be understood in the specification and claims to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which only one member of the group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or the entire group member is present in, used in, or otherwise relevant to a given product or process. Furthermore, the invention should be understood to encompass all modifications, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims that are introduced into other claims dependent on the same base claim (or any other claims that are related), unless otherwise noted or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would result. Where elements are presented as a list (e.g., Markush group or similar format), it should be understood that each subgroup of elements is also disclosed and that any element(s) can be removed from the group. In general, where an invention, or an aspect of an invention, is said to include certain elements, features, etc., it should be understood that certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc. For simplicity, those embodiments have not been expressly and specifically set forth herein in all cases. It should also be understood that any embodiment or aspect of the invention can be expressly excluded from the claims, regardless of whether a specific exclusion is recited in the specification. Publications, websites and other relevant materials mentioned herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference.

Claims

1. 1. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, said composition comprising an antioxidant, and wherein said oxidized IGF-1 species does not increase by more than 50% after three months of storage at a temperature of about 25°C.

2. 2. The composition of claim 1, wherein said oxidized IGF-1 species does not increase by more than 25% after three months of storage at a temperature of about 25°C.

3. 3. The composition of claim 1 or 2, wherein the oxidized IGF-1 species does not increase by more than 10% after 3 months of storage at a temperature of about 25°C.

4. 2. The composition of claim 1, wherein said oxidized IGF-1 species does not increase substantially after three months of storage at a temperature of about 25°C.

5. 1. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in a 1:1 molar ratio, said composition comprising an antioxidant, and wherein the percentage of said oxidized IGF-1 species does not increase by more than 20% after 3 months of storage at a temperature of 2-8°C.

6. 6. The composition of claim 5, wherein said oxidized IGF-1 species does not increase by more than 10% after 3 months of storage at a temperature of 2-8°C.

7. 7. The composition of claim 6, wherein the oxidized IGF-1 species does not increase substantially after 3 months of storage at a temperature in the range of (eg, between) 2-8°C.

8. 2. A composition according to any one of the preceding claims, wherein the antioxidant is methionine, for example at a concentration of 0.2mM to 50mM, such as 0.5mM to 5mM, in particular 0.5mM or 5mM.

9. 9. A composition according to any one of claims 1 to 8, wherein the antioxidant is sodium thiosulfate, for example at a concentration in the range (for example between) 0.2 mM and 50 mM, such as 0.5 mM to 5 mM, in particular 0.5 mM or 5 mM.

10. 2. The composition of any one of the preceding claims, wherein less than 1% of the IGF-1 is present as oxidized species in the protein complex upon 3 months of storage, such as less than 0.6% of the IGF-1 is present as oxidized species in the protein complex upon 3 months of storage.

11. The composition of any one of the preceding claims, wherein the amount of oxidized species is determined by RP-UPLC or SEC-HPLC.

12. 2. The composition according to any one of the preceding claims, wherein the IGFBP-3 comprises less than 5% trisulfide variants, such as less than 2% trisulfide variants, such as less than 1% trisulfide variants, in particular less than 0.5% trisulfide variants.

13. A method for producing an insulin-like growth factor 1 (IGF-1) protein complex, comprising the steps of: providing a composition comprising recombinant IGF-1; adding an antioxidant to said composition; and purifying said IGF-1 from said composition.

14. 24. The method according to claim 23, wherein the antioxidant is methionine, for example added at a concentration of 0.2 mM to 10 mM, such as 0.5 mM to 5 mM, in particular 0.5 mM or 5 mM.

15. 15. The method according to claim 13 or 14, wherein the antioxidant is sodium thiosulfate, for example at a concentration of 0.2 mM to 10 mM, for example 0.5 mM to 5 mM, in particular 0.5 mM or 5 mM.

16. 16. The method of any one of claims 13 to 15, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 50% after 3 months of storage at a temperature of about 25°C, such as does not increase by more than 20%, such as does not increase by more than 10%, in particular does not increase substantially after 3 months of storage at a temperature of about 25°C.

17. 18. The method of any one of claims 13 to 17, wherein the oxidized IGF-1 species in the purified IGF-1 composition does not increase by more than 20% after 3 months of storage at a temperature of 2-8°C, such as does not increase by more than 10%, such as does not increase substantially after 3 months of storage at a temperature of 2-8°C.

18. 18. The method of any one of claims 13 to 17, wherein the purified IGF-1 protein has less than 0.6% oxidized IGF-1 species.

19. The method of any one of claims 13 to 18, wherein the recombinant IGF-1 protein is expressed in E. coli.

20. The method of any one of claims 13 to 19, wherein the recombinant IGF-1 protein is purified from inclusion bodies.

21. 42. The method of any one of claims 23-41, wherein the method further comprises adding insulin-like growth factor binding protein 3 (IGFBP-3) to the IGF-1 protein to form a protein complex.

22. 43. The method of claim 42, wherein the protein complex comprises IGF-1 and IGFBP-3 in a 1:1 molar ratio.

23. 23. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and an insulin-like growth factor binding protein (e.g., IGFBP-3), produced by the method of any one of claims 13-22.

24. A composition comprising an isolated protein complex comprising insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3), e.g., in a 1:1 molar ratio, said composition comprising a bulking agent, a buffer and a surfactant.

25. 25. The composition according to claim 24, wherein the bulking agent is sucrose or trehalose, in particular trehalose.

26. 26. The composition of claim 24 or 25, wherein the buffer is histidine or phosphate.

27. The composition according to any one of claims 24 to 26, wherein the buffer has a pH of 5.5-6.5.

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