(398av) Engineering Studies of the Effect of pH, Temperature and Protein Tertiary Structure on ?-Lactoglobulin a and B Separation in Anion-Exchange Chromatography

In this presentation, we explore the effect of pH and temperature on separating a mixture of similar proteins, namely β-lactoglobulin A (LGA) and β-lactoglobulin B (LGB) in anion-exchange chromatography. The proteins are similar in size and shape, however they carry a slight difference in negative charge at basic pH. In order to separate them, a chromatography column with Sepharose Q Fast Flow was used. The proteins were separated at a range of temperatures and pH values, and the separation factor at each condition was evaluated. Along with that, to get the general trend of protein behavior on an anion exchange resin, bovine serum albumin (BSA) as model protein was run as well. The experimental results were matched to a theoretical model in order to compute the adsorption and desorption rate values of the proteins. The data demonstrated that the separation is improved at increased temperature and pH, and the data was correlated to see how the net charge of the molecule can be used to predict the separation. Furthermore, the tertiary structures of LGA and LGB are analyzed to illustrate the tertiary structure effect on the separation. We observed that LGA has a significant negative charge on the outside of the protein, which causes it to be retained more strongly. Predicting the equilibrium constant allows our theoretical model to predict the elution peak at any pH and temperature condition for this system.