Role of organic modifier and gradient shape in RP-HPLC separation: analysis of GCSF variants.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of therapeutic proteins continues to play a significant role in product characterization. This study focuses on two key aspects of HPLC method development, namely the selection of organic modifier and the gradient shape. Separation of granulocyte colony-stimulating factor variants is being used as a case study to illustrate these concepts. The results demonstrate that careful selection of a binary or ternary mixture of solvents with water is an important factor to be considered for achieving the desired resolution of closely related impurities. The resolution of different types of impurities has been shown to be selective toward the choice of eluent along with the ratio in which they are mixed. In addition, this study also presents a systematic approach for selection of gradient shape based on center point solvent composition, initial solvent composition and the steepness of the gradient. The approach proposed in this study was successfully used to reduce the time of analysis from 70 min for the pertinent European Pharmacopeia method to 15 min by using a solvent system with two organic modifiers (acetonitrile and methanol) along with a sigmoidal-shaped gradient.