Study of relative response factors and mass balance in forced degradation studies with liquid chromatography/photo-diode array detector/evaporative light scattering detector/mass spectrometry system.

A case study was performed using photodiode array detection (PDA) in combination with evaporative light scattering (ELS) detection and mass spectrometry (MS) to assess both mass balance and the relative response factors (RRFs) in the forced degradation studies of the drug substance, glimepiride. The RRF value, which is the ratio of the response factor of the impurity to that of the API, was first determined using calibration curves of standards. This conventional technique was compared to a second, multi-detection technique, which used the PDA and ELS detectors to determine both the ultraviolet (UV) peak area and the concentration (based on measurement by ELS) in a single analysis. The resulting RRF values were then applied to forced degradation studies (acidic hydrolysis and oxidation) of glimepiride drug substance. This analysis was used to assess mass balance as well as impurity quantification. The impact of applying impurity RRF values was evaluated and found to have a significant impact on the percent of impurity quantified and on mass balance calculations at higher degradation levels. In addition, MS was used to quantify a non-chromophoric by-product and assess its impact on mass balance. Analysis of the forced degradation studies by MS detection also provided confirmation of the degradation pathway with new insights. Specifically, MS revealed differences in the stereoselectivity of the degradation processes: the acidic hydrolysis degradation was found to be stereoselective with the formation of the trans by-product while the oxidation degradation produced both the cis/trans isomers in a non-stereoselective reaction.

Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates.

In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.