Size Exclusion Chromatography (SEC) Analysis of Monoclonal Antibodies in Biopharmaceuticals
A technical guide to Size Exclusion Chromatography (SEC) for purity and aggregate analysis of therapeutic monoclonal antibodies.
Size Exclusion Chromatography (SEC) Analysis of Monoclonal Antibodies in Biopharmaceuticals Abstract Size Exclusion Chromatography (SEC) is the primary analytical technique for evaluating the purity, monomer integrity, and aggregation state of therapeutic monoclonal antibodies (mAbs) in biopharmaceutical manufacturing. Introduction Therapeutic proteins and monoclonal antibodies are structurally complex macromolecules sensitive to aggregation and fragmentation during production and storage. Regulatory authorities mandate rigorous aggregate quantification to ensure patient safety and product efficacy [1]. ROWELL supplies biopharmaceutical laboratories with specialized SEC columns designed for high-resolution protein separation. Key Parameters in Biopharmaceutical SEC | Parameter | Technical Consideration | Analytical Objective | | :--- | :--- | :--- | | Pore Size | 200 Å to 300 Å diol-bonded silica or hybrid particles | Optimal size exclusion range for 150 kDa IgG molecules. | | Mobile Phase Buffer | Aqueous phosphate-buffered saline (PBS) with low ionic strength | Prevents electrostatic interactions between proteins and column packing. | | Column Dimensions | 4.6 mm or 7.8 mm ID with 150–300 mm length | Balances high resolution with fast analysis times. | 1. Eliminating Secondary Ionic Interactions Unwanted secondary electrostatic or hydrophobic interactions between proteins and the stationary phase surface cause peak tailing and misquantification of aggregates. Modern SEC phases feature advanced hydrophilic diol coatings to eliminate these artifacts. 2. High-Resolution Aggregate Separation Resolving low levels of high-molecular-weight (HMW) aggregates from the main monomer peak requires high column efficiency and narrow particle size distributions. Practical Recommendat