Dynamic Light Scattering

Dynamic light scattering (DLS) is an additional technique also convenient for the molecular weight evaluation of PEGylated proteins, as it can measure the molecular radii of the samples (Kusterle et al., 2008; Gokarn et al., 2012), and discriminate between linear and branched PEGs (Wan et al., 2017). This method, among others, was used by Vernet et al. in 2016 to assess the first large-scale study with the site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total (Vernet et al., 2016). In addition, Khameneh et al. conducted a study in which site-specific PEGylated hGH was prepared by using microbial transglutaminase. Physicochemical properties, size and zeta potentials of native and PEGylated hGH, were evaluated by DLS, indicating that the size and zeta potentials of the protein were increased and decreased respectively by PEGylation, enhancing the stability of the protein (Khameneh et al., 2016). Recently, Meneguetti et al. applied DLS for the characterization of a novel N-terminal PEGylated asparaginase, showing that the PEGylation of ASNase caused an increase in the hydrodynamic diameter of the protein related to the increase in the amount of PEG attached to the protein (Meneguetti et al., 2019). The DLS approach has been used in the characterization not only of PEGylated proteins, but also of PEGylated organic nanotubes, revealing that PEGylation dramatically improves the dispersibility of the nanotubes in saline buffer (Ding et al., 2014). Despite its wide use in the characterization of the hydrodynamic radius of PEGylated proteins, this methodology presents certain disadvantages in its application, such as the presence of large particles that can also be detected during the analysis; low resolution when the populations are close in size or a highly polydispersed sample; light absorption by the dispersant can interfere with detection because of their viscosity as well as the density of the particles. These are important parameters to take into account when carrying out this type of analysis.