Analysis of PEGylated Protein by Tetra Detection Size Exclusion Chromatography

SEC analysis is important for process development, formulation optimization, release testing, stability, comparability, and forced degradation studies SEC coupled with light scattering (LS), ultra-violent (UV), and refractive index (RI) detection offers detailed molecular weight information about proteins, protein complexes, and glycosylated proteins (1-3). THEORY The light-scattering intensity of polymer molecules in solution is proportional to the molecular weight and concentration of the sample (4): ... where, Rθ is the excess intensity of light scattered at angle θ, C is the sample concentration, Mw is the molecular weight, A^sub 2^ is the second virial coefficient and K is an optical constant, which is proportional to (dn/dC)^sup 2^. Because the concentration of the sample solution in SEC is highly dilute, A2 is often negligible: ... where, P(θ) is the particle scattering function, which represents the angular dependency of light-scattering intensity and is related to radius of gyration (Rg) of the polymer molecule.