Confocal Raman characterization of different protein desorption behaviors from chromatographic particles.

Confocal Raman spectroscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure protein desorption from chromatographic particles. Monoclonal antibody was loaded onto the Fractogel EMD SO3 (M) cation exchanger at either pH 5 or pH 4. Confocal Raman measurement suggests that only the protein loaded at pH 5 is able to release from chromatographic particles in the elution buffer. Detailed comparison of high-quality spectra indicates that, while proteins loaded at both pH values showed a predominant β-sheet conformation, protein loaded at pH 4 has a broader amide I band with more intensity in the >1680 cm(-1) region. This small but clear and reproducible amide I bandwidth increase is not observed for protein in the solution state at pH 4. No definitive assignment of the increased Raman intensity in the >1680 cm(-1) region could be made, but it might be related to structural changes involved in the association of protein molecules in the adsorbed state, which helps to explain the nearly 100% retention under elution conditions of the monoclonal antibody adsorbed at pH 4 in chromatographic particles.