Methods for analyzing goserelin and related products

Abstract

The present application provides methods for analyzing and purifying degarelix or a pharmaceutically acceptable salt thereof containing at least one related impurity. Methods for analyzing and purifying degarelix or a pharmaceutically acceptable salt thereof containing Compound A and / or Compound D as impurities are also provided. Further provided are degarelix or a pharmaceutically acceptable salt thereof prepared and / or selected using the disclosed methods.

Description

[0001] Cross-references to related applications

[0002] This patent application claims the benefit of U.S. Provisional Application No. 62 / 944,276, filed December 5, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background Technology

[0003] Degarelix (e.g., degarelix acetate) is a gonadotropin-releasing hormone (GnRH) used as a hormone therapy for the treatment of prostate cancer. Degarelix (e.g., degarelix acetate) acts immediately and inhibits gonadotropins, testosterone, and prostate-specific antigen (PSA). Degarelix (e.g., degarelix acetate) is a synthetic peptide, usually sold as an acetate salt, N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-4-((S)-dihydroorotamido))-L-phenylalanyl-4-ureido-D-phenylalanyl-L-leucyl-N6-isopropyl-L-lysyl-L-prolyl-D-aminopropionamide.

[0004] Conventional synthetic techniques used to produce degarelk (e.g., degarelk acetate) can produce certain impurities (e.g., compounds AG, I, K, and M) that can be difficult to analyze and / or separate from degarelk (e.g., degarelk acetate). In particular, compounds A and D are not only difficult to separate from degarelk, but also difficult to quantify as individual impurities.

[0005] In view of the foregoing, there is a need for improved methods to analyze each impurity commonly present in degarelk (e.g., degarelk acetate), and preparation techniques to separate impurities (e.g., compounds AG, I, K, and M, and more particularly compounds A and D) from degarelk (e.g., degarelk acetate). Summary of the Invention

[0006] This invention provides a method for analyzing a sample containing degarelk or a pharmaceutically acceptable salt thereof and at least one related impurity. In one aspect, the method includes:

[0007] (a) Elution of a sample by a high-performance liquid chromatography (HPLC) column to produce a chromatogram resolving degarelk or a pharmaceutically acceptable salt thereof and at least one related impurity, wherein the chromatogram includes a first peak with a first area representing degarelk in the sample and a second peak with a second area representing at least one related impurity in the sample.

[0008] (b) Determine the first area under the first peak representing degarelk or its pharmaceutically acceptable salt in the sample.

[0009] (c) determining a second area under a second peak representing the at least one related impurity in the sample, and

[0010] (d) determining the concentration of the at least one related impurity in the sample based on the first area and the second area. The eluting includes eluting the sample with mobile phase A comprising a first aqueous solution having a first pH and a first organic solvent and mobile phase B comprising a second aqueous solution having a second pH and a second organic solvent, wherein the first and second pH values are different. The present invention also provides a batch of degarelix or a pharmaceutically acceptable salt thereof selected based on the results of the method, and the batch thus selected.

[0011] The present invention also provides a method of analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof and Compound A, the method comprising:

[0012] (a) eluting the sample isocratically through a high performance liquid chromatography (HPLC) column with a mobile phase to produce a chromatogram that separates degarelix and Compound A, wherein the chromatogram comprises a first peak representing degarelix in the sample having a first area, and a second peak representing Compound A in the sample having a second area;

[0013] (b) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the sample,

[0014] (c) determining the second area under the second peak representing Compound A in the sample, and

[0015] (d) determining the concentration of Compound A in the sample based on the first area and the second area. The present invention also provides a batch of degarelix or a pharmaceutically acceptable salt thereof selected based on the results of the method, and the batch thus selected.

[0016] The present invention also provides a method of analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof, Compound A and at least one related impurity other than Compound A, the method comprising:

[0017] (a) eluting a first portion of the sample through a high performance liquid chromatography (HPLC) column to produce a first chromatogram of the resolved degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity, the first chromatogram comprising a first peak representing degarelix in the sample having a first area, and a second peak representing at least one related impurity in the sample having a second area, wherein elution of the first portion of the sample comprises elution with mobile phase A and mobile phase B, the mobile phase A comprising a first aqueous solution having a first pH and a first organic solvent, the mobile phase B comprising a second aqueous solution having a second pH and a second organic solvent, wherein the first and second pH values are different, and

[0018] (b) eluting a second portion of the sample through the HPLC column to produce a second chromatogram of the resolved degarelix and compound A, the second chromatogram comprising a first peak representing degarelix in the sample having a first area and a second peak representing compound A in the sample having a second area, wherein elution of the second portion of the sample comprises isocratic elution with the mobile phase,

[0019] (c) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the first chromatogram and the second chromatogram,

[0020] (d) determining the second area under the peak representing the at least one related impurity in the first chromatogram,

[0021] (e) determining the second area under the second peak representing compound A in the second chromatogram, and

[0022] (f) determining the concentration of the at least one related impurity and compound A in the sample based on the first and second areas of the first and second chromatograms. The present invention also provides a batch of degarelix or a pharmaceutically acceptable salt thereof selected based on the results of this method, and the batch thus selected.

[0023] The present invention also provides a method of purifying degarelix or a pharmaceutically acceptable salt thereof and compound A, the method comprising:

[0024] (a) eluting degarelix through a chromatography column with a mobile phase to separate degarelix or a pharmaceutically acceptable salt thereof containing compound A to produce a purified form of degarelix or a pharmaceutically acceptable salt thereof, and

[0025] (b) isolating the purified degarelix or a pharmaceutically acceptable salt thereof, wherein elution comprises isocratic elution of degarelix from the chromatography column with the mobile phase. The present invention further provides a purified form of degarelix or a pharmaceutically acceptable salt thereof prepared by this and other methods described herein.

[0026] BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A high performance liquid chromatogram showing a split solution analyzed according to an exemplary method.

[0028] Figure 2 A high performance liquid chromatogram showing a synthesis sample analyzed according to an exemplary method.

[0029] Figure 3 A high performance liquid chromatogram showing a split solution prepared and analyzed according to an exemplary method.

[0030] Figure 4 A high performance liquid chromatogram showing a synthesis sample analyzed according to an exemplary method. DETAILED DESCRIPTION

[0032] The present invention provides methods for analyzing, isolating, and / or purifying degarelix or a pharmaceutically acceptable salt thereof (e.g., degarelix acetate) that contains certain impurities (e.g., certain impurities identified as Compounds A-G, I, K, and M). As used herein, the term "degarelix" refers to N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L- serine-4-((S)-dihydroorotamido)-L-phenylalanyl-4-ureido-D-phenylalanyl-L-leucyl-N6- isopropyl-L-lysyl-L-prolyl-D-alaninamide. The degarelix to be analyzed and / or purified according to the present invention can be in its free base form or can exist as a pharmaceutically acceptable salt thereof. In certain embodiments, the degarelix described herein is in the form of degarelix acetate.

[0033] As used herein, the phrase "pharmaceutically acceptable salt" is intended to include salts derived from the parent compound containing a basic or acidic moiety. Generally, such salts can be prepared by reacting free acid or base forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. For example, inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), organic acids (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methanesulfonic acid, or benzylsulfonic acid), inorganic bases (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), organic bases (e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl) aminomethane, guanidine, choline, or cinchonidine), or amino acids (e.g., lysine, arginine, or alanine) can be used. Generally, nonaqueous media like diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66:2-19 (1977).

[0034] Impurities of degarelix can be synthetic intermediates, synthetic byproducts, degradation products, or aggregation products. For example, impurities associated with degarelix can include impurities identified as Compounds A-G, I, K, and M, as described herein, the structures of which are provided in Table 1.

[0035] Table 1. Degarelix impurities

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] In one aspect, the present application provides a method of analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity (e.g., one or more of Compounds B, C, D, E, F, G, I, K, and M), the method comprising: (a) passing the sample through a high pressure liquid chromatography (HPLC) column (e.g., a WATERS Sunfire C18 column, 4.6 x 50 mm, 3.5 micron, 100 A, 5 pm / min, 40 °C, 1 mL / min, 220 nm); and (b) detecting the presence of the at least one related impurity.TM XSelect CSH C18 3.5 μιη, 4.6 x 150 mm P / N 18600527) to produce a chromatogram of the resolved degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity, wherein the chromatogram includes a first peak having a first area representing degarelix in the sample and a second peak having a second area representing the at least one related impurity in the sample; (b) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the sample, (c) determining the second area under the second peak representing the at least one related impurity in the sample, and (d) determining the concentration of the at least one related impurity in the sample based on the first area and the second area, wherein eluting includes eluting the sample with mobile phase A and mobile phase B, the mobile phase A including a first aqueous solution having a first pH and a first organic solvent, the mobile phase B including a second aqueous solution having a second pH and a second organic solvent, wherein the first pH and the second pH are different. In certain embodiments, the at least one related impurity includes Compound D.

[0043] The sample analyzed can include any suitable composition containing degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity (e.g., Compounds B, C, D, E, F, G, I, K, and M). In some embodiments, the sample for analysis is an aqueous solution comprising degarelix or a pharmaceutically acceptable salt thereof. The aqueous solution can also include one or more polar organic solvents, such as acetonitrile, methanol, ethanol, (iso)propanol, dimethylformamide, diethyl ether, tetrahydrofuran, ethyl acetate, and the like. Alternatively or additionally, the aqueous solution can further include one or more inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), organic acids (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), inorganic bases (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), or organic bases (e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, guanidine, choline, or cinchonidine), and the like. In certain embodiments, the sample comprises degarelix or a pharmaceutically acceptable salt thereof, at least one related impurity (e.g., one or more of Compounds B, C, D, E, F, G, I, K, and M), water, acetonitrile, and acetic acid.

[0044] The sample for analysis can have any suitable amount of degarelix or a pharmaceutically acceptable salt thereof. For example, the sample for analysis can comprise degarelix at a concentration of about 0.01 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.25 mg / mL to about 2.5 mg / mL, or about 0.4 mg / mL to about 1 mg / mL.

[0045] In embodiments in which the sample for analysis comprises acetic acid, the sample can have any suitable amount of acetic acid. For example, the sample for analysis can be applied to the column as a solution comprising acetic acid at a volume concentration of 0.01-1%, a volume concentration of 0.05-0.5%, or a volume concentration of about 0.1%.

[0046] The sample for analysis can have any suitable amount of polar organic solvent. For example, the sample for analysis can be applied to the column as a solution comprising a polar solvent at a volume concentration of about 1-50%, a volume concentration of about 1-40%, a volume concentration of about 1-30%, a volume concentration of about 5-50%, a volume concentration of about 5-40%, a volume concentration of about 5-30%, a volume concentration of about 10-50%, a volume concentration of about 10-40%, or a volume concentration of about 10-30%. In certain embodiments, the sample for analysis comprises a polar solvent at a volume concentration of about 20%.

[0047] Chromatograms generated according to the present application can be generated by detecting ultraviolet (UV) absorption at any wavelength suitable for detecting degarelix. In some embodiments, the chromatogram is generated by detecting ultraviolet (UV) absorption at about 245 nm of degarelix and at least one related impurity eluted from the column. The concentration of the at least one related impurity in the sample can be determined based on the first area and the second area in the generated chromatogram using one or more techniques known to those skilled in the art.

[0048] The HPLC column used according to the present application can be any suitable HPLC column comprising a chromatographic resin for reverse phase chromatography. As described herein, “reverse phase chromatography” refers to any chromatographic technique for analyzing and / or separating sample materials using a hydrophobic stationary phase and an aqueous phase or a hydrophilic mobile phase. Thus, the chromatographic resins described herein are generally hydrophobic chromatographic resins. For example, the chromatographic resin can be octadecyl carbon chain (C18) bonded silica, C8 bonded silica, pure silica, cyano bonded silica, phenyl bonded silica, activated carbon, or a combination thereof. The HPLC column described herein can have any suitable dimensions and any suitable particle size. In certain embodiments, the HPLC column is a BEH C18 column (e.g., BEH C18 1.7 μιη, 2.1 x 50 mm P / N 186003593) available from WATERS TM a commercially available C18 XSelect column (e.g., XSelect CSH C18 3.5 μιη, 4.6 x 150 mm P / N 186005270).

[0049] The sample can be eluted through the HPLC column at any suitable temperature. For example, elution can occur at a temperature from about 10 °C to about 50 °C, from about 20 °C to about 40 °C, or from about 25 °C to about 35 °C. In some embodiments, elution of the sample through the HPLC column can occur at a temperature of about 25 °C, about 30 °C, or about 35 °C.

[0050] The sample can be eluted through the HPLC column at any suitable flow rate. For example, elution can occur at a flow rate of about 0.1 mL / min to about 10 mL / min, from about 0.5 mL / min or about 5 mL / min, or from about 0.5 mL / min to about 2 mL / min. In some embodiments, the flow rate of the sample through the HPLC column can be about 0.5 mL / min, about 0.6 mL / min, 0.7 mL / min, about 0.8 mL / min, 0.9 mL / min, about 1 mL / min, 1.1 mL / min, about 1.2 mL / min, 1.3 mL / min, about 1.4 mL / min, or 1.5 mL / min.

[0051] The run time of the sample eluted through the HPLC column can include any suitable length of time, including run times commonly used in reverse phase HPLC separations. For example, the run time can be from about 5 minutes to about 5 hours, from about 15 minutes to about 4 hours, from about 30 minutes to about 2 hours, or from about 45 minutes to about 1.5 hours. In some embodiments, the run time is about 30 minutes, about 45 minutes, about 1 hour, about 75 minutes, or about 90 minutes.

[0052] In some embodiments, eluting the sample through the HPLC column includes eluting the sample with mobile phase A and mobile phase B, wherein mobile phase A comprises a first aqueous solution having a first pH and a first organic solvent, and mobile phase B comprises a second aqueous solution having a second pH and a second organic solvent, wherein the first pH value and the second pH value are different. The difference between the first pH value and the second pH value can be at least about 0.2 pH units, at least about 0.5 pH units, at least about 1 pH unit, at least about 1.5 pH units, or at least about 2 pH units. In certain embodiments, the difference between the first pH value and the second pH value is about 2 pH units. For example, in some embodiments, the first pH is about 5.5 and the second pH is about 3.5.

[0053] The first aqueous solution can have any suitable pH that is different from the second pH. For example, the first pH can be from about 3 to about 9, from about 4 to about 8, from about 5 to about 7, or from about 5 to about 6. In some embodiments, the pH of the first aqueous solution is about 4.5, about 5, about 5.5, about 6, or about 6.5. For example, in some embodiments, the pH of the first aqueous solution is about 5.5.

[0054] The second aqueous solution can have any suitable pH that is different from the first pH. For example, the second pH can be from about 1 to about 7, from about 2 to about 6, from about 3 to about 5, or from about 3 to about 4. In some embodiments, the pH of the second aqueous solution is about 2.5, about 3, about 3.5, about 4, or about 4.5. For example, in some embodiments, the pH of the second aqueous solution is about 3.5.

[0055] The first and second aqueous solutions can be buffered using any suitable buffer, which can be, for example, in the form of a salt. For example, the first and second aqueous solutions can each independently be buffered using a suitable salt, including a phosphate salt, a citrate salt, a formate salt, an acetate salt, or a combination thereof. In certain embodiments, the first and second aqueous solutions each comprise a phosphate buffer. For example, the first and second aqueous solutions can each include potassium phosphate.

[0056] The first and second aqueous solutions can each comprise any suitable amount of the buffer. For example, the first and second aqueous solutions can each independently comprise from about 1 mM to about 500 mM of the buffer salt (e.g., potassium phosphate), from about 1 mM to about 100 mM of the buffer salt (e.g., potassium phosphate), or from about 1 mM to about 50 mM of the buffer salt (e.g., potassium phosphate). In certain embodiments, the first and second aqueous solutions can each independently comprise about 10 mM of the buffer salt (e.g., potassium phosphate), about 15 mM of the buffer salt (e.g., potassium phosphate), about 20 mM of the buffer salt (e.g., potassium phosphate), about 25 mM of the buffer salt (e.g., potassium phosphate), about 30 mM of the buffer salt (e.g., potassium phosphate), about 35 mM of the buffer salt (e.g., potassium phosphate), or about 40 mM of the buffer salt (e.g., potassium phosphate).

[0057] The mobile phase A and the mobile phase B can each comprise a first organic solvent and a second organic solvent, respectively. The first and second organic solvents can each independently be selected from any suitable organic solvent, including organic solvents commonly used for HPLC separations. In some embodiments, the first and second organic solvents are at least partially miscible with water. For example, the first and second organic solvents can each independently comprise acetonitrile, methanol, ethanol, (iso)propanol, dimethylformamide, diethyl ether, tetrahydrofuran, ethyl acetate, or a combination thereof. In certain embodiments, the mobile phase A and the mobile phase B comprise the same organic solvent. For example, in some embodiments, the first and second organic solvents of the mobile phase A and the mobile phase B, respectively, comprise acetonitrile.

[0058] The mobile phase A and the mobile phase B can each independently comprise any suitable amount of the first organic solvent and the second organic solvent. For example, the mobile phase A and B can each independently comprise about 1-50% by volume, about 1-40% by volume, about 1-30% by volume, about 5-50% by volume, about 5-40% by volume, about 5-30% by volume, about 10-50% by volume, about 10-40% by volume, or about 10-30% by volume of the first organic solvent and the second organic solvent. In some embodiments, the mobile phase A comprises about 25% by volume of the first organic solvent. In other embodiments, the mobile phase B comprises about 35% by volume of the first organic solvent.

[0059] In certain embodiments, the mobile phase A comprises a 25 mM potassium phosphate solution at a pH of 5.50 and acetonitrile in a ratio of about 75:25 (solution:acetonitrile).

[0060] In certain embodiments, the mobile phase B comprises a 25 mM potassium phosphate solution at a pH of 3.50 and acetonitrile in a ratio of about 65:35 (solution:acetonitrile).

[0061] In one exemplary embodiment of the present application, the mobile phase A comprises a 25 mM potassium phosphate solution at a pH of 5.50 and acetonitrile in a ratio of about 75:25 (solution:acetonitrile), and the mobile phase B comprises a 25 mM potassium phosphate solution at a pH of 3.50 and acetonitrile in a ratio of about 65:35 (solution:acetonitrile).

[0062] Eluting the sample through the HPLC column can comprise using a gradient of the mobile phase A and the mobile phase B. The gradient can transition, for example, from the mobile phase A to the mobile phase B, and / or from the mobile phase B to the mobile phase A. An exemplary gradient for use in at least this aspect of the present application is as follows:

[0063] Time (min) Mobile phase A Mobile phase B 0 100 0 5 100 0 45 0 100 46 100 0 60 100 0

[0064] In some embodiments, the methods of the present application further comprise selecting a batch of degarelix or a pharmaceutically acceptable salt thereof, e.g., for use in preparing a pharmaceutical composition for therapeutic administration to a subject in need thereof, based on the determination of the concentration of at least one relevant impurity in a batch sample analyzed according to the present application. Without wishing to be bound by any particular theory, it is believed that the present application provides an improved method of analyzing, quantifying, and / or isolating impurities related to and / or structurally similar to degarelix or a pharmaceutically acceptable salt thereof, resulting in improved quality control of batch selection and / or purification of degarelix and its salts. In certain embodiments, the present application further provides compositions of degarelix or a pharmaceutically acceptable salt thereof selected by the methods described herein.

[0065] In another aspect, the present application provides a method for analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof and Compound A, the method comprising: (a) passing the sample through a high performance liquid chromatography (HPLC) column (e.g., a PHENOMENEX® Gemini NX-C18, 4.6 x 150 mm, 3 μιη P / N 00F-4453-E0) to produce a chromatogram resolving degarelix and Compound A, wherein the chromatogram comprises a first peak having a first area representing degarelix in the sample and a second peak having a second area representing Compound A in the sample; (b) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the sample, (c) determining the second area under the second peak representing Compound A in the sample, and (d) determining the concentration of Compound A in the sample based on the first and second areas, wherein eluting comprises isocratically eluting the sample with a mobile phase. TM Gemini NX-C18,4.6x150mm,3μm P / N00F-4453-E0) to produce a chromatogram resolving degarelix and Compound A, wherein the chromatogram comprises a first peak having a first area representing degarelix in the sample and a second peak having a second area representing Compound A in the sample; (b) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the sample, (c) determining the second area under the second peak representing Compound A in the sample, and (d) determining the concentration of Compound A in the sample based on the first and second areas, wherein eluting comprises isocratically eluting the sample with a mobile phase.

[0066] The sample analyzed can comprise any suitable composition containing degarelix or a pharmaceutically acceptable salt thereof and Compound A. In some embodiments, the sample for analysis is an aqueous solution comprising degarelix or a pharmaceutically acceptable salt thereof. The aqueous solution can further comprise one or more polar organic solvents, such as acetonitrile, methanol, ethanol, (iso)propanol, dimethylformamide, diethyl ether, tetrahydrofuran, ethyl acetate, and the like. Alternatively or additionally, the aqueous solution can further comprise one or more inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), organic acids (such as oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), inorganic bases (such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), or organic bases (such as methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)aminomethane, guanidine, choline, or cinchonidine), and the like. In certain embodiments, the sample comprises degarelix or a pharmaceutically acceptable salt thereof, Compound A, water, acetonitrile, and acetic acid.

[0067] The sample for analysis can have any suitable amount of degarelix or a pharmaceutically acceptable salt thereof. For example, the sample for analysis can comprise degarelix at a concentration of about 0.01 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.25 mg / mL to about 2.5 mg / mL, or about 0.4 mg / mL to about 1 mg / mL.

[0068] In embodiments where the sample for analysis comprises acetic acid, the sample can comprise any suitable amount of acetic acid. For example, the sample for analysis can be applied to the column as a solution comprising acetic acid at a volume concentration of 0.01-1%, a volume concentration of 0.05-0.5%, or a volume concentration of about 0.1%.

[0069] The sample for analysis can also include any suitable amount of polar organic solvent. For example, the sample for analysis can be applied to the column as a solution comprising a polar solvent at a volume concentration of about 1-50%, a volume concentration of about 1-40%, a volume concentration of about 1-30%, a volume concentration of about 5-50% volume, a volume concentration of about 5-40% volume, a volume concentration of about 5-30% volume, a volume concentration of about 10-50% volume, a volume concentration of about 10-40% volume, or a volume concentration of about 10-30%. In certain embodiments, the sample for analysis comprises a polar solvent at a volume concentration of about 20%.

[0070] The chromatogram can be generated by detecting ultraviolet (UV) absorption at any wavelength suitable for detecting degarelix. In some embodiments, the chromatogram is generated by detecting ultraviolet (UV) absorption at about 245 nm of degarelix and Compound A eluted from the column. The concentration of Compound A in the sample can be determined based on the first and second areas in the generated chromatogram using one or more techniques known to one of skill in the art.

[0071] The HPLC column can be any suitable HPLC column comprising a chromatographic resin for reversed-phase chromatography. For example, the chromatographic resin can be octadecyl carbon chain (C18) bonded silica, C8 bonded silica, plain silica, cyano bonded silica, phenyl bonded silica, activated carbon, or a combination thereof. The HPLC column described herein can have any suitable dimensions and any suitable particle size. In certain embodiments, the HPLC column for use in the second aspect is a KINETEX™ EVO C18 column (e.g., KINETEX™ EVO C18, 4.6 x 150 mm, 2.6 μιη P / N 00A-4403-E0) available from PHENOMENEX®. TM a commercially available Gemini NX-C18 column (e.g., Gemini NX-C18, 4.6 x 150 mm, 3 μιη P / N 00F-4453-E0).

[0072] Elution of the sample through the HPLC column can occur at any suitable temperature. For example, elution can occur at a temperature of about 10 °C to about 50 °C, about 20 °C to about 40 °C, or about 20 °C to about 30 °C. In some embodiments, elution of the sample through the HPLC column can occur at a temperature of about 20 °C, about 25 °C, or about 30 °C.

[0073] Elution of the sample through the HPLC column can occur at any suitable flow rate. For example, elution can occur at a flow rate of about 0.1 mL / min to about 10 mL / min, about 0.5 mL / min to about 5 mL / min, or about 0.5 mL / min to about 2 mL / min. In some embodiments, elution of the sample through the HPLC column occurs at a flow rate of about 0.5 mL / min, about 0.6 mL / min, 0.7 mL / min, about 0.8 mL / min, 0.9 mL / min, about 1 mL / min, 1.1 mL / min, about 1.2 mL / min, 1.3 mL / min, about 1.4 mL / min, or 1.5 mL / min.

[0074] The run time for elution of the sample through the HPLC column can be any suitable length of time, including run times typically used for reverse phase HPLC separations. For example, the run time can be about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, about 30 minutes to about 2 hours, or about 45 minutes to about 1.5 hours. In some embodiments, the run time is about 15 minutes, 30 minutes, about 45 minutes, or about 1 hour.

[0075] In some embodiments, elution of the sample through the HPLC column includes isocratic elution of the sample with a mobile phase. As used herein, the term “isocratic” or “isocratic elution” refers to a process in which a single mobile phase is used to elute the sample, i.e., the mobile phase remains constant or unchanging.

[0076] The mobile phase can include an aqueous solution and an organic solvent.

[0077] The aqueous solution of the mobile phase can have any suitable pH. For example, the pH of the aqueous solution of the mobile phase can be about 6 to about 13, about 7 to about 12, about 8 to about 11, or about 9 to about 11. In some embodiments, the pH of the aqueous solution of the mobile phase is about 9, about 9.5, about 10, about 10.5, or about 11. In certain embodiments, the pH of the aqueous solution of the mobile phase is about 10.

[0078] Any suitable buffering agent can be used to buffer the aqueous solution of the mobile phase, which can be in the form of a salt, for example. For example, a salt comprising a phosphate, a citrate, a formate, an acetate, or a combination thereof can be used to buffer the aqueous solution of the mobile phase. In certain embodiments, the aqueous solution of the mobile phase comprises an acetate buffer. For example, the aqueous solution of the mobile phase can include ammonium acetate.

[0079] The aqueous solution of the mobile phase can include any suitable amount of a buffer. For example, the aqueous solution of the mobile phase can include about 1 mM to about 500 mM of a buffer salt (e.g., ammonium acetate), about 1 mM to about 250 mM of a buffer salt (e.g., ammonium acetate), or about 1 mM to about 100 mM of a buffer salt (e.g., ammonium acetate). In certain embodiments, the mobile phase can include about 30 mM of a buffer salt (e.g., ammonium acetate), about 35 mM of a buffer salt (e.g., ammonium acetate), about 40 mM of a buffer salt (e.g., ammonium acetate), about 45 mM of a buffer salt (e.g., ammonium acetate), about 50 mM of a buffer salt (e.g., ammonium acetate), about 55 mM of a buffer salt (e.g., ammonium acetate), or about 60 mM of a buffer salt (e.g., ammonium acetate).

[0080] The mobile phase can also include an organic solvent. The organic solvent of the mobile phase can be selected from any suitable organic solvent. In some embodiments, the organic solvent is at least partially miscible with water. For example, the organic solvent can include acetonitrile, methanol, ethanol, (iso)propanol, dimethylformamide, diethyl ether, tetrahydrofuran, ethyl acetate, or a combination thereof. In certain embodiments, the organic solvent of the mobile phase includes acetonitrile.

[0081] The mobile phase can include any suitable amount of the organic solvent. For example, the mobile phase can include about 1-50% by volume, about 1-40% by volume, about 1-30% by volume, about 5-50% by volume, about 5-40% by volume, about 5-30% by volume, about 10-50% by volume, about 10-40% by volume, or about 20-40% by volume of the organic solvent. In certain embodiments, the mobile phase includes about 35% by volume of the organic solvent.

[0082] In some embodiments, the mobile phase includes an ammonium acetate buffer solution and acetonitrile. In certain embodiments, the ammonium acetate buffer solution includes about 1 mM to about 100 mM of ammonium acetate (e.g., about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, or about 60 mM) and has a pH of about 9 to about 11 (e.g., about 9, about 9.5, about 10, about 10.5, or about 11). In some embodiments, the ammonium acetate buffer solution includes about 45 mM of ammonium acetate, has a pH of about 10, and the ratio of the ammonium acetate buffer solution to acetonitrile in the mobile phase is about 65:35.

[0083] In some embodiments, the methods of the present application further comprise selecting a batch of degarelix or a pharmaceutically acceptable salt thereof, e.g., for use in preparing a pharmaceutical composition for therapeutic administration to a subject in need thereof, based on the determination of the concentration of Compound A in a batch sample analyzed according to the present application. Without wishing to be bound by any particular theory, it is believed that the present application provides an improved method of analyzing, quantifying, and / or isolating impurities related to and / or structurally similar to degarelix or a pharmaceutically acceptable salt thereof that cannot otherwise be adequately separated and / or distinguished from degarelix and / or each other, thereby improving the quality control of batch selection. More specifically, it is believed that the present application provides an improved method to effectively isolate Compound A from degarelix or a salt thereof and / or quantify Compound A in a sample comprising degarelix or a salt thereof. In certain embodiments, the present application further provides a batch of degarelix or a pharmaceutically acceptable salt thereof selected according to the present application.

[0084] In another aspect, the present application provides a method of analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof, Compound A, and at least one related impurity other than Compound A, the method comprising: eluting a first portion of the sample through a high performance liquid chromatography (HPLC) column (e.g., WATERS TM XSelect CSH C18 3.5μm,4.6x150mm P / N 186005270) to produce a first chromatogram that resolves degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity other than Compound A (e.g., Compounds B, C, D, E, F, G, I, K, and M), the first chromatogram comprising a first peak having a first area representing degarelix in the sample, and a second peak having a second area representing at least one related impurity in the sample, wherein elution of the first portion of the sample comprises elution with mobile phase A and mobile phase B, the mobile phase A comprising a first aqueous solution having a first pH and a first organic solvent, the mobile phase B comprising a second aqueous solution having a second pH and a second organic solvent, wherein the first pH value and the second pH value are different, and (b) eluting a second portion of the sample through the HPLC column (e.g., PHENOMENEX TMa second portion of the sample is eluted to produce a second chromatogram of resolved degarelix and Compound A, the second chromatogram comprising a first peak having a first area representing degarelix in the sample, and a second peak having a second area representing Compound A in the sample, wherein the eluting of the second portion of the sample comprises isocratic elution with the mobile phase, (c) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the first and second chromatograms, (d) determining the second area under the peak representing at least one related impurity in the first chromatogram, (e) determining the second area under the second peak representing Compound A in the second chromatogram, and (f) determining the concentrations of at least one related impurity and Compound A in the sample based on the first and second areas of the first and second chromatograms.

[0085] This aspect of the application can be combined with any particular embodiment of the other aspects of the application described herein to determine the amount of Compound A and / or to isolate Compound A and at least one related impurity other than Compound A. In certain embodiments, the at least one related impurity other than Compound A comprises Compound D. Without wishing to be bound by any particular theory, it is believed that Compound A and D are uniquely challenging to separate and quantify by conventional methods due to their structural similarity (e.g., structural or electronic). However, utilizing the methods of the application comprising elution with mobile phases A and B as described herein allows one to quantify Compound D in addition to quantifying other related impurities, and utilizing the methods of the application comprising resolving degarelix and Compound A as described herein allows one to quantify Compound A. Thus, the application allows one to readily isolate and / or quantify Compound A and D in a batch of degarelix or a salt thereof. Accordingly, the application provides purified degarelix or a pharmaceutically acceptable salt thereof comprising 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.%, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound A and no more than 0.3 wt.% (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound D relative to degarelix or a pharmaceutically acceptable salt thereof. In certain embodiments, the application provides purified degarelix or a pharmaceutically acceptable salt thereof comprising 0.2 wt.% or less of Compound A and 0.15 wt.% or less of Compound D relative to degarelix or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the present application further comprises selecting a batch of degarelix or a pharmaceutically acceptable salt thereof, e.g., for use in preparing a pharmaceutical composition for therapeutic administration to a subject in need thereof, based on the determination of the concentration of at least one related impurity other than Compound A (e.g., Compound D) and Compound A. Without wishing to be bound by any particular theory, it is believed that the present application provides an improved method of analyzing, and / or quantifying, degarelix or a pharmaceutically acceptable salt thereof containing Compound A and at least one other related impurity, e.g., Compound D, thereby resulting in improved quality control of batch selection, particularly by providing an improved method of separating and / or quantifying Compound D and Compound A in a batch of degarelix. In certain embodiments, the present application also provides a batch of degarelix or a pharmaceutically acceptable salt thereof selected by the method.

[0087] In some embodiments, a batch of degarelix or a pharmaceutically acceptable salt thereof selected according to the present application comprises 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound A and 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound D, relative to degarelix or a pharmaceutically acceptable salt thereof. In certain embodiments, a batch of degarelix or a pharmaceutically acceptable salt thereof selected by the method of the present application comprises 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound A and 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound D, relative to degarelix or a pharmaceutically acceptable salt thereof, after the batch is stored at about 2-8 °C for about 24 months.

[0088] In yet another aspect, the present application provides a method of purifying degarelix or a pharmaceutically acceptable salt thereof and Compound A, the method comprising: (a) separating degarelix or a pharmaceutically acceptable salt thereof and Compound A by eluting a sample through a chromatographic column with a mobile phase, thereby producing a purified material comprising degarelix or a pharmaceutically acceptable salt thereof, wherein eluting comprises eluting the sample from the chromatographic column with the mobile phase in an isocratic manner.

[0089] The chromatography column used in the purification methods of the present application can be any suitable column comprising a chromatography resin for reverse phase chromatography. For example, the chromatography resin can be octadecyl carbon chain (C18) bonded silica, C8 bonded silica, plain silica, cyano bonded silica, phenyl bonded silica, activated carbon, or combinations thereof. In some embodiments, the chromatography column is a preparative high performance liquid chromatography (HPLC) column. In certain embodiments, the chromatography column is a C18 column having an average particle size of about 2 pm to about 20 pm (e.g., about 2 pm to about 10 pm or about 2 pm to about 5 pm) and a pore size of about 100 A to about 300 A (e.g., about 100 A to about 200 A or about 100 A to about 150 A). In some embodiments, the chromatography column is a C18 column having an average particle size of about 5 pm to about 10 pm and a pore size of about 100 A to about 200 A. In certain embodiments, the chromatography column is a C18 column having an average particle size of about 5 pm and a pore size of about 100 A. to about (e.g., about about or about ) silicone grafted C18 resin.

[0090] The mobile phase used in the purification methods of the present application can comprise an aqueous solution and an organic solvent.

[0091] The aqueous solution of the mobile phase used in the purification methods of the present application can have any suitable pH. For example, the pH of the aqueous solution of the mobile phase can be about 6 to about 13, about 7 to about 12, about 8 to about 11, or about 9 to about 11. In some embodiments, the pH of the pH mobile phase aqueous solution is about 9, about 9.5, about 10, about 10.5, or about 11. In certain embodiments, the pH of the mobile phase aqueous solution is about 10.

[0092] The aqueous solution of the mobile phase in the purification methods of the present application can be buffered using any suitable reagent, for example, can be in the form of a salt for buffering the solution. For example, the aqueous solution of the mobile phase can be buffered using a salt comprising a phosphate, citrate, formate, acetate, or combinations thereof. In certain embodiments, the aqueous solution of the mobile phase comprises an acetate buffer. In some embodiments, the aqueous solution of the mobile phase comprises ammonium acetate.

[0093] The aqueous solution of the mobile phase of the purification methods of the present application can comprise any suitable amount of the buffering agent. For example, the aqueous solution of the mobile phase can comprise about 1 mM to about 500 mM of the buffering salt (e.g., ammonium acetate), about 1 mM to about 250 mM of the buffering salt (e.g., ammonium acetate), or about 1 mM to about 100 mM of the buffering salt (e.g., ammonium acetate). In certain embodiments, the mobile phase can comprise about 30 mM of the buffering salt (e.g., ammonium acetate), about 35 mM of the buffering salt (e.g., ammonium acetate), about 40 mM of the buffering salt (e.g., ammonium acetate), about 45 mM of the buffering salt (e.g., ammonium acetate), about 50 mM of the buffering salt (e.g., ammonium acetate), about 55 mM of the buffering salt (e.g., ammonium acetate), or about 60 mM of the buffering salt (e.g., ammonium acetate).

[0094] The mobile phase of the purification method of the present application can also include an organic solvent. The organic solvent of the mobile phase can be selected from any suitable organic solvent, including organic solvents typically used for reverse phase HPLC separations. In some embodiments, the organic solvent is at least partially miscible with water. For example, the organic solvent can include acetonitrile, methanol, ethanol, (iso)propanol, dimethylformamide, diethyl ether, tetrahydrofuran, ethyl acetate, or combinations thereof. In certain embodiments, the organic solvent of the mobile phase comprises acetonitrile.

[0095] The mobile phase of the purification method of the present application can include any suitable amount of organic solvent. For example, the mobile phase can include about 1-50% by volume, about 1-40% by volume, about 1-30% by volume, about 5-50% by volume, about 5-40% by volume, about 5-30% by volume, about 10-50% by volume, about 10-40% by volume, or about 20-40% by volume of the organic solvent. In certain embodiments, the mobile phase includes about 35% by volume of the organic solvent.

[0096] In some embodiments, the mobile phase used in the purification method of the present application includes an ammonium acetate buffer solution and acetonitrile. In certain embodiments, the ammonium acetate buffer solution includes about 1 mM to about 100 mM ammonium acetate (e.g., about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, or about 60 mM) and has a pH of about 9 to about 11 (e.g., about 9, about 9.5, about 10, about 10.5, or about 11). In some embodiments, the ammonium acetate buffer solution includes about 45 mM ammonium acetate, has a pH of about 10, and the ratio of the ammonium acetate buffer solution to acetonitrile in the mobile phase is about 65:35.

[0097] The purified degarelix or pharmaceutically acceptable salt thereof obtained by the purification method of the present application preferably comprises 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound A relative to degarelix or a pharmaceutically acceptable salt thereof. Accordingly, the present application also provides purified degarelix or a pharmaceutically acceptable salt thereof comprising 0.3 wt.% or less (e.g., 0.2 wt.% or less, 0.15 wt.% or less, 0.1 wt.% or less, or 0.01 wt.% or less) of Compound A relative to degarelix or a pharmaceutically acceptable salt thereof. The present application further provides a pharmaceutical composition comprising an excipient and purified degarelix or a pharmaceutically acceptable salt thereof comprising 1.0 wt.% or less (e.g., 0.5 wt.% or less, 0.3 wt.% or less, 0.2 wt.% or less, or 0.1 wt.% or less) of Compound A relative to degarelix or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition comprising an excipient and purified degarelix or a pharmaceutically acceptable salt thereof comprises 1.0 wt.% or less (e.g., 0.5 wt.% or less, 0.3 wt.% or less, 0.2 wt.% or less, or 0.1 wt.% or less) of Compound A and 1.0 wt.% or less (e.g., 0.5 wt.% or less, 0.3 wt.% or less, 0.2 wt.% or less, or 0.1 wt.% or less) of Compound D relative to degarelix or a pharmaceutically acceptable salt thereof after storage of the pharmaceutical composition at about 2-8 °C for about 24 months. The pharmaceutical composition of the present application preferably includes a therapeutically effective amount of purified degarelix or a pharmaceutically acceptable salt thereof produced according to the present application.

[0098] The pharmaceutical composition can be a liquid or a solid, such as a lyophilized solid. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. For example, the pharmaceutical composition can comprise one or more excipients that can serve to protect degarelix or a pharmaceutically acceptable salt thereof during manufacture and / or storage, such as a sugar, an amino acid, a polymer, a surfactant, a buffer, an antioxidant, or a preservative. In some embodiments, the pharmaceutical composition can include pharmaceutically acceptable auxiliary substances as required to make a physiologically acceptable formulation, such as pH adjusting and buffering agents, tonicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, mannitol, and the like. The concentration of purified degarelix or a pharmaceutically acceptable salt thereof in these formulations can vary as will be appreciated by those of ordinary skill in the art and can be selected based on, for example, fluid volumes, viscosities, body weights, and the like, consistent with the particular mode of administration and the needs of the patient. Examples

[0099] Aspects of the subject matter described herein, including embodiments, can be beneficially used alone or in combination with one or more other aspects or embodiments. Without limitation to the foregoing description, certain non-limiting examples of the present application are provided below. As will be apparent to one of ordinary skill in the art upon reading the present disclosure, each individually numbered aspect or embodiment can be used or combined with any other individually numbered aspect disclosed herein to provide support for all such combinations of aspects, but is not limited to the combinations of aspects or embodiments expressly provided below:

[0100] 1. A method for analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity, the method comprising:

[0101] (a) eluting the sample through a high pressure liquid chromatography (HPLC) column to produce a chromatogram resolving degarelix or a pharmaceutically acceptable salt thereof and the at least one related impurity, wherein the chromatogram comprises a first peak having a first area representing degarelix in the sample, and a second peak having a second area representing the at least one related impurity in the sample;

[0102] (b) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the sample,

[0103] (c) determining the second area under the second peak representing the at least one related impurity in the sample, and

[0104] (d) determining the concentration of the at least one related impurity in the sample based on the first area and the second area,

[0105] wherein eluting comprises eluting the sample with a mobile phase A and a mobile phase B, the mobile phase A comprising a first aqueous solution having a first pH and a first organic solvent, and the mobile phase B comprising a second aqueous solution having a second pH and a second organic solvent, wherein the first pH value and the second pH value are different.

[0106] 2. The method according to embodiment 1, wherein the difference between the first pH value and the second pH value is at least about 1 pH unit.

[0107] 3. The method according to embodiment 1 or embodiment 2, wherein the first pH is about 5.5 and the second pH is about 3.5.

[0108] 4. The method according to any one of embodiments 1-3, wherein the first and second aqueous solutions of the mobile phase A and the mobile phase B, respectively, comprise a phosphate buffer.

[0109] 5. The method according to any one of embodiments 1-4, wherein the first and second organic solvents of the mobile phase A and the mobile phase B, respectively, comprise acetonitrile.

[0110] 6. The method of any one of embodiments 1-5, wherein the mobile phase A comprises a 25 mM potassium phosphate solution at pH 5.50 and acetonitrile in a ratio of about 75:25 (solution:acetonitrile).

[0111] 7. The method of any one of embodiments 1-6, wherein the mobile phase B comprises a 25 mM potassium phosphate solution at pH 3.50 and acetonitrile in a ratio of about 65:35 (solution:acetonitrile).

[0112] 8. The method of any one of embodiments 1-7, wherein elution is performed using a gradient from mobile phase A to mobile phase B according to the following conditions:

[0113] Time (min) Mobile phase A Mobile phase B 0 100 0 5 100 0 45 0 100 46 100 0 60 100 0

[0114] 9. The method of any one of embodiments 1-8, wherein the sample is applied to the column as a solution comprising a volume concentration of 0.01-1% acetic acid.

[0115] 10. The method of any one of embodiments 1-9, wherein a chromatogram is generated by detecting ultraviolet (UV) absorption at about 245 nm of degarelix and at least one related impurity eluted from the column.

[0116] 11. The method of any one of embodiments 1-10, wherein the at least one related impurity comprises Compound B, C, D, E, F, G, I, K, or M.

[0117] 12. The method of any one of embodiments 1-11, wherein the sample comprises degarelix acetate.

[0118] 13. The method of any one of embodiments 1-12, further comprising selecting a batch of degarelix or a pharmaceutically acceptable salt thereof for therapeutic administration to a subject in need thereof based on the determination of the concentration of the at least one related impurity.

[0119] 14. A batch of degarelix or a pharmaceutically acceptable salt thereof selected by the method of embodiment 13.

[0120] 15. A method for analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof and Compound A, the method comprising:

[0121] (a) eluting the sample through a high performance liquid chromatography (HPLC) column to generate a chromatogram resolving degarelix and Compound A, wherein the chromatogram comprises a first peak having a first area representing degarelix in the sample and a second peak having a second area representing Compound A in the sample;

[0122] (b) determining a first area under a first peak in the sample representing degarelix or a pharmaceutically acceptable salt thereof,

[0123] (c) determining a second area under a second peak representing Compound A in the sample, and

[0124] (d) determining a concentration of Compound A in the sample based on the first area and the second area,

[0125] wherein eluting comprises eluting the sample with a mobile phase isocratic.

[0126] 16. The method of embodiment 15, wherein the mobile phase comprises an ammonium acetate buffer solution and acetonitrile.

[0127] 17. The method of embodiment 16 or embodiment 17, wherein the ammonium acetate buffer solution comprises 45 mM ammonium acetate and has a pH of about 10.0.

[0128] 18. The method of any one of embodiments 15-17, wherein the ratio of ammonium acetate buffer solution to acetonitrile in the mobile phase is about 65:35.

[0129] 19. The method of any one of embodiments 15-18, wherein the sample is applied to the column as a solution comprising a volume concentration of 0.01-1% acetic acid.

[0130] 20. The method of any one of embodiments 15-19, wherein the chromatogram is produced by detecting ultraviolet (UV) absorption at about 245 nm of degarelix and Compound A eluted from the column.

[0131] 21. The method of any one of embodiments 15-20, wherein the sample comprises degarelix acetate.

[0132] 22. The method of any one of embodiments 15-21, further comprising selecting a batch of degarelix or a pharmaceutically acceptable salt thereof for therapeutic administration to a subject in need thereof based on the determination of the concentration of Compound A.

[0133] 23. A batch of degarelix or a pharmaceutically acceptable salt thereof selected by the method of embodiment 22.

[0134] 24. A method of analyzing a sample comprising degarelix or a pharmaceutically acceptable salt thereof, Compound A, and at least one related impurity other than Compound A, the method comprising:

[0135] (a) eluting a first portion of the sample through a high performance liquid chromatography (HPLC) column to produce a first chromatogram of the resolved degarelix or a pharmaceutically acceptable salt thereof and at least one related impurity, the first chromatogram comprising a first peak representing degarelix in the sample having a first area, and a second peak representing at least one related impurity in the sample having a second area, wherein elution of the first portion of the sample comprises elution with mobile phase A, the mobile phase A comprising a first aqueous solution having a first pH and a first organic solvent, and mobile phase B comprises a second aqueous solution having a second pH and a second organic solvent, wherein the first pH value and the second pH value are different, and

[0136] (b) eluting a second portion of the sample through the HPLC column to produce a second chromatogram of the resolved degarelix and Compound A, the second chromatogram comprising a first peak representing degarelix in the sample having a first area, and a second peak representing Compound A in the sample having a second area, wherein elution of the second portion of the sample comprises elution with mobile phase isocratic,

[0137] (c) determining the first area under the first peak representing degarelix or a pharmaceutically acceptable salt thereof in the first chromatogram and the second chromatogram,

[0138] (d) determining the second area under the peak representing the at least one related impurity in the first chromatogram,

[0139] (e) determining the second area under the second peak representing Compound A in the second chromatogram, and

[0140] (f) determining the concentration of the at least one related impurity and Compound A in the sample based on the first area and the second area of the first chromatogram and the second chromatogram.

[0141] 25. The method according to embodiment 24, wherein the at least one related impurity comprises Compound D.

[0142] 26. The method according to embodiment 24 or embodiment 25, wherein the sample comprises degarelix acetate.

[0143] 27. The method according to any one of embodiments 24-26, further comprising selecting a batch of degarelix or a pharmaceutically acceptable salt thereof for therapeutic administration to a subject in need based on the determination of the concentration of the at least one related impurity and Compound A.

[0144] 28. A batch of degarelix or a pharmaceutically acceptable salt thereof selected by the method of embodiment 27.

[0145] 29. The batch of embodiment 28, wherein the batch comprises 0.3 wt.% or less of Compound A and 0.3 wt.% or less of Compound D relative to the degarelix or the pharmaceutically acceptable salt thereof after the batch is stored at about 2-8 °C for about 24 months.

[0146] 30. The batch of embodiment 28 or embodiment 29, wherein the batch comprises 0.3 wt.% or less of Compound A and 0.3 wt.% or less of Compound D relative to the degarelix or the pharmaceutically acceptable salt thereof.

[0147] 31. A method of purifying degarelix or a pharmaceutically acceptable salt thereof containing Compound A, the method comprising:

[0148] (a) separating the degarelix or the pharmaceutically acceptable salt thereof and Compound A by eluting the degarelix through a chromatographic column with a mobile phase to produce a purified form of the degarelix or the pharmaceutically acceptable salt thereof, and

[0149] (b) isolating the purified degarelix or the pharmaceutically acceptable salt thereof,

[0150] wherein eluting comprises isocratically eluting the sample from the chromatographic column with the mobile phase.

[0151] 32. The method of embodiment 31, wherein the chromatographic column is a preparative high performance liquid chromatography (HPLC) column.

[0152] 33. The method of embodiment 31 or embodiment 32, wherein the chromatographic column is a silicone-grafted C18 resin having an average particle size of about 2 pm to about 20 pm and a pore size of about to about .

[0153] 34. The method of any one of embodiments 31-33, wherein the mobile phase comprises an ammonium acetate buffer solution and acetonitrile.

[0154] 35. The method of any one of embodiments 31-34, wherein the ammonium acetate buffer solution comprises 45 mM ammonium acetate and has a pH of about 10.0.

[0155] 36. The method of any one of embodiments 31-35, wherein the ratio of the ammonium acetate buffer solution to acetonitrile in the mobile phase is about 65:35.

[0156] 37. The method of any one of embodiments 31-36, wherein the degarelix is applied to the chromatographic column as a solution comprising a volume concentration of 0.01-1% acetic acid.

[0157] 38. The method of any one of embodiments 31-37, wherein the degarelix comprises degarelix acetate.

[0158] 39. The method of any one of embodiments 31-38, wherein the purified degarelix, or a pharmaceutically acceptable salt thereof, comprises 0.3 wt.% or less of Compound A relative to degarelix, or a pharmaceutically acceptable salt thereof.

[0159] 40. The method of any one of embodiments 31-39, wherein the purified degarelix, or a pharmaceutically acceptable salt thereof, comprises 0.2 wt.% or less of Compound A relative to degarelix, or a pharmaceutically acceptable salt thereof.

[0160] 41. The method of any one of embodiments 31-40, wherein the purified degarelix, or a pharmaceutically acceptable salt thereof, comprises 0.1 wt.% or less of Compound A relative to degarelix, or a pharmaceutically acceptable salt thereof.

[0161] 42. Purified degarelix prepared according to embodiment 39.

[0162] 43. Purified degarelix prepared according to embodiment 40.

[0163] 44. Purified degarelix prepared according to embodiment 41.

[0164] 45. A pharmaceutical composition comprising a carrier and the purified degarelix, or a pharmaceutically acceptable salt thereof, of any one of embodiments 42-44.

[0165] 46. Purified degarelix, or a pharmaceutically acceptable salt thereof, comprising 0.3 wt.% or less of Compound A and 0.3 wt.% or less of Compound D relative to degarelix, or a pharmaceutically acceptable salt thereof.

[0166] 47. The purified degarelix of embodiment 46 comprising 0.2 wt.% or less of Compound A and 0.15 wt.% or less of Compound D relative to degarelix, or a pharmaceutically acceptable salt thereof.

[0167] The following examples further illustrate the application but, of course, should not be construed as in any way limiting its scope.

[0168] Example 1

[0169] The following examples describe methods of analyzing and / or isolating certain impurities (e.g., Compound I, Compound M, Compound E, Compound K, Compound D, Compound G, Compound B, and / or Compound C) from degarelix.

[0170] The split solution is prepared by accurately weighing about 27.6 mg of degarelix raw material (equivalent to about 24 mg of degarelix after correction for moisture and acetic acid content) and transferring it to a 50 mL volumetric flask. The degarelix material used should contain about 0.10 wt.% of Compound M, 0.25 to 0.35 wt.% of Compound E, 0.10 to 0.25 wt.% of Compound K, and 0.25 wt.% of Compound A. These impurities can be added to the split solution in the appropriate amounts to achieve the appropriate concentrations. The resulting solution is diluted (using 0.1% acetic acid in 20% acetonitrile in water) to about 0.48 mg / mL of degarelix and about 0.10 wt.% Compound M, 0.25 to 0.35 wt.% Compound E, 0.10 to 0.25 wt.% Compound K, and 0.25 wt.% Compound A.

[0171] The split solution is analyzed by high performance liquid chromatography (HPLC) using the following parameters and separated according to Table 2 using a linear gradient:

[0172] Mobile Phase A: Phosphate Buffer pH 5.5: Acetonitrile (75:25)

[0173] Mobile Phase B: Phosphate Buffer pH 3.5: Acetonitrile (65:35)

[0174] Column: WATERS TM XSelect CSH C18 3.5 μm, 4.6 x 150 mm P / N 186005270

[0175] Column Temperature: 30.0 ± 3.0 °C

[0176] Flow Rate: 1.0 mL / min.

[0177] Injection Volume: 10 μL

[0178] Auto-sampler Temperature: 5 ± 3 °C

[0179] Detection: UV 245 nm

[0180] Run Time: 60 minutes

[0181] Separation Mode: Gradient

[0182] Table 2. Gradient Conditions

[0183] Time (min) Mobile phase A Mobile phase B 0 100 0 5 100 0 45 0 100 46 100 0 60 100 0

[0184] From Figure 1The results shown clearly demonstrate that the above method adequately resolves Compound I, Compound M, Compound E, Compound K, Compound D, Compound G, Compound B, and Compound C from degarelix. Compound A elutes with degarelix, making it impossible to distinguish the amount of degarelix at this stage.

[0185] A synthetic sample of degarelix was analyzed using the above HPLC parameters, and the results are shown in Figure 2 . As shown in Figure 2 the above HPLC parameters adequately resolved Compound I, Compound M, Compound E, Compound K, Compound D, Compound G, Compound B, and Compound C from degarelix obtained from the synthetic sample. The relative amounts of each impurity (Not More Than = NMT) are listed in Table 3.

[0186] Table 3. Impurity specifications

[0187] Compound Amount (wt. %) A Not measured B NMT 0.15 C NMT 0.15 D NMT 0.15 E NMT 0.2 F NMT 0.15 G NMT 0.15 I NMT 0.15 K NMT 0.15 M NMT 0.15 All other NMT 0.1

[0188] Example 2

[0189] The following example shows an exemplary protocol for analyzing and / or separating Compound A from degarelix.

[0190] A resolution solution was prepared by accurately weighing about 27.6 mg of degarelix raw material (equivalent to about 24 mg of degarelix after correction for moisture and acetic acid content) and transferring it to a 50 mL volumetric flask. The degarelix material used should contain about 0.10 wt.% of Compound M, 0.25 wt.% to 0.35 wt.% of Compound E, 0.10 wt.% to 0.25 wt.% of Compound K, and 0.25 wt.% of Compound A. These impurities can be added in the appropriate amounts to the resolution solution to achieve the proper concentrations. The resulting solution was diluted (using 0.1% acetic acid, 20% acetonitrile in water) to about 0.48 mg / mL of degarelix and about 0.10 wt.% of Compound M, 0.25 wt.% to 0.35 wt.% of Compound E, 0.10 wt.% to 0.25 wt.% of Compound K, and 0.25 wt.% of Compound A.

[0191] The resolution solution was analyzed by high performance liquid chromatography (HPLC) using the following parameters, and an isocratic gradient was used for separation:

[0192] Mobile Phase A: 65:35

[0193] 45 mM ammonium acetate buffer pH 10.0: acetonitrile

[0194] Column: PHENOMENEX TMGemini NX-C18, 4.6 x 150 mm, 3 μm P / N 00F-4453-E0

[0195] Column Temperature: 25 ± 3 °C

[0196] Flow Rate: 0.7 mL / min.

[0197] Injection Volume: 10 μL

[0198] Auto-sampler Temperature: 5 ± 3 °C

[0199] Detection: UV 245 nm

[0200] Run Time: 30 minutes

[0201] Separation Mode: Isocratic

[0202] The results from Figure 3 clearly show that the above HPLC parameters adequately resolve Compound A from degarelix.

[0203] To further demonstrate the effectiveness of the method, a synthetic sample of degarelix was analyzed using the above HPLC parameters, and the results are shown in Figure 4 . As shown in Figure 4 , the above HPLC parameters adequately resolved Compound A from degarelix obtained from the synthetic sample. The relative amount of Compound A was no more than (NMT) 0.2 wt. %.

[0204] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0205] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item from the list A or B or any combination of two or more of the listed items A and B, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0206] Preferred embodiments of this application are described herein, including the best mode known to the inventors of practicing the application. Variations of those preferred embodiments are readily apparent to those skilled in the art in view of the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the application to include all such modifications and equivalents that fall within the scope of the claimed subject matter. Additionally, the inventors intend for the application to include any and all combinations of one or more of the foregoing claimed elements.

Claims

1. A method of purifying degarelix or a pharmaceutically acceptable salt thereof containing Compound A, the method comprising: (a) separating degarelix or a pharmaceutically acceptable salt thereof and Compound A by eluting degarelix through a chromatographic column with a mobile phase to produce a purified form of degarelix or a pharmaceutically acceptable salt thereof, and (b) isolating the purified degarelix or a pharmaceutically acceptable salt thereof, wherein the eluting comprises isocratically eluting the sample from the chromatographic column with a mobile phase, wherein the mobile phase is ammonium acetate buffer solution at a pH of about 10.0 and acetonitrile in a ratio of about 65:35; Compound A is D-amino propionamide, N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-serine-4-([2-(5-hydrazinyl)]-acetylamino-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-proline; the chromatographic column is a silicone-grafted C18 resin.

2. The method of claim 1, wherein the chromatographic column has an average particle size of 2 μm to 20 μm and a pore size of 100 Å to 120 Å.

3. The method of claim 1, wherein degarelix is applied to the chromatographic column as a solution comprising a volume concentration of 0.01-1% acetic acid.

4. The method of claim 1, wherein the purified degarelix or a pharmaceutically acceptable salt thereof comprises 0.3 wt.% or less of Compound A relative to degarelix or a pharmaceutically acceptable salt thereof.

5. The method of claim 1, wherein the purified degarelix or a pharmaceutically acceptable salt thereof comprises 0.2 wt.% or less of Compound A relative to degarelix or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the purified degarelix or a pharmaceutically acceptable salt thereof is contained in a pharmaceutical composition.

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