Understanding Hydrophobic Effects in Preparative Chromatography

The effect of varying concentrations of both ethanol and potassium chloride (KCl), as well as temperature, on the retention of three insulin variants, on two hydrophobic interaction chromatography (HIC) and two reversed-phase chromatography (RPC) adsorbents, has been studied experimentally. Both the linear adsorption range and protein load levels on the preparative scale are covered.

The results for the HIC adsobents indicated additional phenomena, with fronting peaks at low load and both fronting and tailing ones at high load, such as double-layer formation or conformational changes. Due to these complications, the HIC systems were not further investigated, while a dynamic model structure was developed for the RPC systems. Data from a solubility study for one of the insulins showed that changes in the activity coefficient of the insulins in the mobile phase cannot alone account for the effects of ethanol. An adsorption mechanism assuming that the adsorption sites are initially occupied by ethanol which is displaced upon adsorption of the insulin gave good agreement with the experimental data.

The resulting model structure is being implemented into a framework for dynamic simulations of chromatographic runs within the whole adsorption range. A description of the temperature effect is to be added, and the final model will be used for a design study using different methods for optimization and robustness analysis.

Preparative chromatography is a separation method which is widely used within the pharmaceutical industry. Many of today's pharamceuticals are based on proteins, and these are often produced by genetically modified microorganisms, e.g. bacteria or yeast. However, the microorganisms also produce a lot of other proteins and substances that can be toxic to humans. Therefore, it is important to purify the protein that is to be used in the final drug.

Chromatography is based on the different tendency of compounds, e.g. proteins, to be in the solid or the liquid phase of the chromatographic system. The compound mix is added to the liquid phase, which is constantly flowing through the porous solid phase, and some compounds stick to the solid phase for a while and come out after a longer time period, while others flow straight through. By collecting the liquid phase exiting the system in different containers, the different compounds can be separated.

Two types of preparative chromatography are based on how fatty the compounds are. Those that are more fatty stick harder to the very fatty solid phase. The tendency of the compounds to stick can be adjusted by adding different chemicals to the liquid phase, making it more or less fatty. In this case, we have studied what happens if different amounts of ethanol and a salt are added. We also tested the effect of the temperature in the system.

Based on the results from the experiments, we made a computer model that can be used to predict what will happen at different temperatures and concetnrations of ethanol and salt. This model will be used to find the combination that gives the largest amouint of protein, that is pure enough, in the most economical way. Hopefully, this model can be used as a tool to design new processes for production of pharmaceuticals in a safer, faster and cheaper way.