Unspecific interactions in different batch variations of agarose based base matrices at different NaCl concentrations.

Abstract: Size exclusion chromatography (SEC) separates molecules based on their actual size rather than their molecular weight. This indicates that the smaller a molecule is, the longer it will be held in the column since it can penetrate the pores more effectively than a bigger molecule, resulting in a longer retention time, whereas the larger molecule has a shorter retention time. In order to achieve reliable and accurate results the asymmetry factor of the packed columns should be between 0.6-1.4. The aim for this project was to examine whether or not the batch variations of the base matrix used to derive WorkBeads™ 40S and 40Q displayed either hydrophobic or hydrophilic interactions by running different types of proteins through columns packed with the base matrices. The project was performed using an ÄKTA explorer equipped with an ultraviolet (UV) detector and a refractive index detector (RID). The data was gathered and analyzed with the Unicorn™ by Cytiva. The results from the first experiments showed that lysozyme did not elute as expected or not at all, thus leading to a concern that the there might be some hydrophobic interactions in the base matrix, which is a porous media in the form of spherical particles that have been selected for their physical stability and inertness (lack of reactivity and adsorptive properties), and lysozyme. With this suspicion in mind, the different batches of the base matrix underwent hydrophobic interaction chromatography (HIC), where the results may be interpreted in a way that there might electrostatic interactions instead of hydrophobic interactions. However, due to the gel not being suitable for HIC the results were unreliable. By subsequently running lysozyme and other proteins through the columns at different NaCl concentrations the results showed consistent elution at NaCl concentrations > 150 mM, yet inconsistent at a concentration of 150 mM for lysozyme. The elution order by size showed that although lysozyme has a larger hydrodynamic radius (Rh) than cytochrome c it eluted later, which is theoretically incorrect, but it might be owing to some of the base matrix's characteristics or the lysozyme's dual nature of expressing both hydrophobic and hydrophilic interactions on the base matrix. Because of the inconsistent results from lysozyme, another experiment just at 150 mM was conducted where lysozyme did elute consistently with a KD value < 1.  Lastly, a titration was performed on the base matrix where some of it was brominated and some of it was not, which was the reference. The results showed that there could be hydrophobic interactions on the brominated sample and hydrophilic interactions on the reference sample. However, what is more likely is that lysozyme is an unreliable protein to use to determine hydrophobicity on this type of gel.