Capillary gel electrophoresis for precise protein quantitation

Abstract

CGE, also known as SDS-CGE, is being established in the pharmaceutical industry replacing SDS-PAGE. In most cases, the method is applied for the identity and purity control of proteins, for example monoclonal antibodies. In order to quantify these components with sufficient precision using the same quality control method, a RSD for the quantitative analysis under 2% is required. A reliable and highly precise CGE method could be obtained after thorough optimization. It was crucial to increase the sample concentration and the injection volume in order to achieve sufficiently high S/N ratios (>70). The application of hydrodynamic injection is beneficial for the precision of the method compared to the traditionally used electrokinetic one. Linearity was demonstrated and LOD and LOQ were estimated. Both injection modes were compared in long series runs (n = 48). Furthermore, the use of an internal standard was investigated. Thus, the RSD% of the migration time was reduced from 0.9 to 0.2% and the RSD% of peak areas was greatly improved. However, the normalization to the total area further reduced the influence of the injection error. RSD% for the peak area ratios of typically between 1 and 2% was provided.