(304b) Adsorption Mechanism of Biopharmaceuticals Onto RPLC Columns

Reversed-phase liquid chromatography (RPLC) is a widely employed purification method for hydrophobic small molecules and biopharmaceuticals since the invention of the first hydrocarbon polymer-coated material in 1971 [1]. This technique is preferred over other chromatographic modes as the resolution and selectivity obtained from this technique are sufficient to separate complex mixtures. This benefit is particularly true for biopharmaceuticals, as RPLC can separate homologous peptide sequences despite their subtle hydrophobicity and chemical differences.

Despite many years of this technique being used for analytical and preparative applications, method development is still based on trial-and-error approaches. This approach is due to the plethora of commercial columns available in the market as well as a lack of understanding of the fundamentals of adsorption relevant to these columns. The measurement of excess adsorption isotherm helps to describe the adsorption behaviour of an adsorbate molecule, such as a peptide (analyte), interacting with the surface adsorption sites on the adsorbent (stationary phase). In this type of isotherm, as the concentration of the adsorbate increases, the amount of adsorbate adsorbed onto the surface also increases until the surface becomes saturated, indicating full coverage of a monolayer or multilayer of the adsorbate. This method can also be used to understand the chemical interactions between the mobile phase and the stationary phase without the presence of an analyte. These types of experiments are essential for interpreting and predicting the adsorption retention mechanisms as well as the physicochemical properties of the RPLC column.

In this study, we studied the intrinsic differences between commercial RPLC columns measured through excess isotherms. A total of 10 columns with different particle sizes (5 and 10 µm), phases (C8, C18 and phenyl-hexyl) and application types (analytical and preparative) were tested to measure the adsorption of ethanol, methanol and acetonitrile when used as mobile phase. Our results have shown that each combination of columns and mobile phases produces a different thickness of the adsorption layer, meaning that the adsorption of an analyte is not only dependent on the hydrophobicity of the stationary phase but also on the available space created by the thickness of the layer. This is quite relevant for larger molecules, such as biopharmaceuticals, as the lack of available adsorption space on the surface can significantly reduce the performance of the column. Additionally, we have estimated the hydrophobic heterogeneity as a property of the stationary phase surface when mobile phases with different solvent strengths are adsorbed onto the surface. We have found that the solvent strength of acetonitrile and particle size of 5 µm produces a 4-fold increase in apparent hydrophobic surface (acetonitrile/water adsorbed) compared to 10 µm in combination with methanol as organic solvent.