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].