Development and partial validation of a SEC method for high-resolution separation and quantifi cation of monoclonal antibodies

Size exclusion chromatography (SEC) is an important technique for the characterization of high molecular substances such as proteins or polymers. It is the standard method to determine and quantify aggregation levels for protein-based pharmaceuticals and is generally required for regulatory approval. This Application Note describes a simple and sensitive method for the quantitative analysis of monoclonal antibodies (mAbs) by SEC. Separation and quantifi cation of mAbs was achieved by the Agilent 1260 Bio-inert Infi nity Quaternary LC System using an Agilent Bio SEC-3 column. Bio SEC-3 columns are a breakthrough technology for size exclusion chromatography. They are packed with spherical, narrowly dispersed 3-μm silica particles coated with a proprietary hydrophilic layer. Calibration of the column was performed using protein molecular weight markers. Robustness of the method for routine analysis was established by validation studies using monoclonal antibody. Furthermore, the Bio SEC-3 column was able to separate and detect mAb aggregates. The bio-inertness and corrosion resistance of the instrument coupled with a simple and reproducible method is a practical solution for routine QA/QC analysis of monoclonal antibody for the biopharmaceutical industry. Author M. Sundaram Palaniswamy Agilent Technologies, Inc. Bangalore, India