Methods for identification and quantitation of abaloparatide and related peptide impurities

A method of providing abaloparatide in a form substantially free of beta-Asp10 abaloparatide, the method comprising:

• (i) providing a sample from a batch of abaloparatide;
• (ii) introducing the sample to an ultra-high performance chromatography (UPLC) system comprising a column having an interior for accepting a stationary phase, and an immobilized stationary phase within said interior of the column, wherein the immobilized stationary phase comprises C18-modified particles;
• (iii) flowing the sample through the immobilized stationary phase for a period of time with an eluent comprising a first mobile phase comprising aqueous perchloric acid and a second mobile phase which is acetonitrile, thereby eluting components of the sample from the immobilized stationary phase in the eluant;
• (iv) detecting abaloparatide and beta-Asp10 abaloparatide in the sample using a detector;
• (v) quantifying the amount of beta-Asp10 abaloparatide present relative to abaloparatide;
• (vi) ascertaining that the amount beta-Asp10 abaloparatide is not more than 1% by weight based on the weight of abaloparatide;
• and
• (vii) approving release of the batch of abaloparatide.

A method of manufacturing an abaloparatide drug product suitable for subcutaneous administration, the method comprising:

• (i) analyzing a sample from a batch of abaloparatide drug product, wherein analyzing comprises:
• a) introducing the sample to an ultra-high performance chromatography (UPLC) system comprising a column having an interior for accepting a stationary phase, and an immobilized stationary phase within said interior of the column, wherein the immobilized stationary phase comprises C18-modified particles;
• b) flowing the sample through the immobilized stationary phase for a period of time with an eluent comprising a first mobile phase comprising aqueous perchloric acid and a second mobile phase which is acetonitrile, thereby eluting components of the sample from the immobilized stationary phase in the eluant;
• c) detecting and quantifying any isomeric and degradation impurities present in the sample using a detector;
• ii) determining the abaloparatide is substantially free of isomeric and degradation impurities;
• and
• iii) approving the release of the batch of abaloparatide.

A chromatographic method of analyzing a sample of abaloparatide, the method comprising:

• (vi) providing a sample comprising abaloparatide;
• (vii) introducing the sample to an ultra-performance chromatography (UPLC) system comprising a column having an interior for accepting a stationary phase, and an immobilized stationary phase within said interior of the column, wherein the immobilized stationary phase comprises C18-modified particles;
• (viii) flowing the sample through the immobilized stationary phase for a period of time with an eluant comprising a first mobile phase comprising water, formic acid, and heptafluorobutyric acid, and a second mobile phase comprising acetonitrile, formic acid and heptafluorobutyric acid, thereby eluting the abaloparatide from the immobilized stationary phase in the eluant;
• (ix) detecting the abaloparatide present in the eluant using a detector;
• and
• (x) quantifying the abaloparatide present in the sample.

A method of providing abaloparatide in a form substantially free of beta-Asp10 abaloparatide, the method comprising:

• (i) providing a sample from a batch of abaloparatide;
• (ii) introducing the sample to an ultra-high performance chromatography (UPLC) system comprising a column having an interior for accepting a stationary phase, and an immobilized stationary phase within said interior of the column, wherein the immobilized stationary phase comprises C18-modified particles;
• (iii) flowing the sample through the immobilized stationary phase for a period of time with an eluant comprising a first mobile phase comprising water, formic acid, and heptafluorobutyric acid, and a second mobile phase comprising acetonitrile, formic acid and heptafluorobutyric acid, thereby eluting components of the sample from the immobilized stationary phase in the eluant;
• (iv) detecting abaloparatide and beta-Asp10 abaloparatide in the sample using a detector;
• (v) quantifying the amount of beta-Asp10 abaloparatide present relative to abaloparatide;
• (vi) ascertaining that the amount beta-Asp10 abaloparatide is not more than 1% by weight based on the weight of abaloparatide;
• and
• (vii) approving release of the batch of abaloparatide.

A method of manufacturing an abaloparatide drug product suitable for subcutaneous administration, the method comprising:

• (ii) analyzing a sample from a batch of abaloparatide drug product, wherein analyzing comprises:
• d) introducing the sample to an ultra-high performance chromatography (UPLC) system comprising a column having an interior for accepting a stationary phase, and an immobilized stationary phase within said interior of the column, wherein the immobilized stationary phase comprises C18-modified particles;
• e) flowing the sample through the immobilized stationary phase for a period of time with an eluant comprising a first mobile phase comprising water, formic acid, and heptafluorobutyric acid, and a second mobile phase comprising acetonitrile, formic acid and heptafluorobutyric acid, thereby eluting components of the sample from the immobilized stationary phase in the eluant;
• f) detecting and quantifying any isomeric and degradation impurities present in the sample using a detector;
• iv) determining the abaloparatide is substantially free of isomeric and degradation impurities;
• and
• v) approving the release of the batch of abaloparatide.