Hydrophobicity analysis of Chinese Hamster Ovary host cell proteins using analytical hydrophobic interaction chromatography and LC-MS.

The surface hydrophobicity of the host cell proteins (HCP) present in Chinese Hamster Ovary (CHO) cell cultures can have a significant effect on the design and performance of the downstream process. This study examined the use of analytical hydrophobic interaction chromatography (HIC) in combination with mass spectrometry (LC-MS) to probe the surface hydrophobicity of the broad array of HCP present in CHO cell cultures. The HCP covered a range of hydrophobicity, with good reproducibility between cell culture samples. The GRAVY number was ineffective at predicting the hydrophobicity of the individual HCP, showing almost no correlation with the elution time measured during HIC. Detailed analysis of 17 selected HCP showed better results using the residue accessibility and the area of the largest hydrophobic patch, both of which evaluate the hydrophobicity by considering only the amino acid residues located on the protein surface as determined directly from the three-dimensional protein structures calculated by AlphaFold. These results highlight the potential use of analytical HIC coupled with LC-MS to evaluate the surface hydrophobicity of complex protein mixtures as well as the limitations of current methods for predicting protein hydrophobicity based on the amino acid composition and protein structure.