3.1.2 Size exclusion chromatography
Proteins in Size exclusion chromatography (SEC) are separated based on the size in a porous matrix, useful for diverse protein conditions and purifications [17]. SEC employs materials like dextran, reducing salt and determining molecular weights, using materials like Sephadex G type, polyacrylamide, or agarose [15]. Conventional SEC columns are valuable for proteins like erythropoietin and monoclonal antibodies (mAbs) in high-performance size exclusion chromatography (HP-SEC), assessing glycoprotein quality [18]. SEC-HPLC/UV efficiently monitors protein monomers, aggregates, and nitration degrees (NDs), directly quantifying bovine serum albumin (BSA) [20].
High-molecular-weight aggregates, significant in mAb and bispecific mAb production, increase immunogenicity. Silica-coated SEC columns with hydrophobic silica particles enhance reliability in identifying key protein aggregates [19]. Recent research combines UHPLC-SEC with techniques like UV-MALS-QELS-RI, accurately measuring molar mass for biotherapeutic proteins [21]. SDS-PAGE, Fast-SEC, and DLS analyze 18 kDa heat shock proteins, assessing their potential for generating aggregates under various conditions. MALS-UV-RI/SEC-HPLC distinguishes aggregation levels in protein antigens for a Clostridium difficile vaccine, a protein-based vaccine [19].