exenatide and bivalirudin

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed using three peptide drugs: salmon calcitonin, bivalirudin, and exenatide as model systems to assess the suitability of this approach for monitoring peptide drug product quality. Calcitonin and its related impurities displayed linear responses over the range from 0.1 to 10 μM (R (2) values for calcitonin salmon, Glu(14)-calcitonin, and acetyl-calcitonin were 0.995, 0.996, and 0.993, respectively). Intra-assay precision in terms of relative standard deviation (%RSD) was less than 10% at all tested concentrations. The accuracy of the method was greater than 85% as measured by spiking 0.1, 0.3, and 1% of Glu(14)-calcitonin and acetyl-calcitonin into a stock calcitonin solution. Limits of detection for calcitonin, Glu(14)-calcitonin, and acetyl-calcitonin were 0.02, 0.03, and 0.04 μM, respectively, indicating that an impurity present at less than 0.1% (0.1 μM) of the drug product API concentration (107 μM) could be detected. Method validation studies analyzing bivalirudin and exenatide drug products exhibited similar results to calcitonin salmon in regard to high selectivity, sensitivity, precision, and linearity. Added benefits of using LC-HRMS-based methods are the ability to also determine amino acid composition, confirm peptide sequence, and quantify impurities, even when they are co-eluting, within a single experiment. LC-HRMS represents a promising approach for the quality control of peptides including the measurement of any peptide-related impurities. While the development work performed here is focus on peptide drug products, the principles could be adapted to peptide drug substance.

Topics: Amino Acid Sequence; Calcitonin; Chromatography, Liquid; Exenatide; Hirudins; Limit of Detection; Mass Spectrometry; Peptide Fragments; Peptides; Quality Control; Recombinant Proteins; Venoms

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described.

Topics: Countercurrent Distribution; Exenatide; Hirudins; Magnetic Resonance Spectroscopy; Peptide Fragments; Peptides; Recombinant Proteins; Solubility; Solvents; Venoms