EMD Millipore , 80 Ashby Road, Bedford, Massachusetts 01730, United States.
Anal Chem. 2014 Jan 21;86(2):1007-15. doi: 10.1021/ac401081s. Epub 2014 Jan 9.
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.
共焦拉曼光谱学是一种非破坏性分析技术，它将振动光谱的化学信息与共焦显微镜的空间分辨率相结合。它首次被应用于测量蛋白质从色谱颗粒上的解吸。单克隆抗体在 pH 值为 5 或 pH 值为 4 时被加载到 Fractogel EMD SO3（M）阳离子交换剂上。共焦拉曼测量表明，只有在 pH 值为 5 时加载的蛋白质能够在洗脱缓冲液中从色谱颗粒中释放出来。对高质量光谱的详细比较表明，虽然在两种 pH 值下加载的蛋白质都显示出主要的β-折叠构象，但在 pH 值为 4 时加载的蛋白质在>1680 cm（-1）区域具有更宽的酰胺 I 带，强度更大。在 pH 值为 4 的溶液状态下，蛋白质没有观察到这种小但清晰且可重复的酰胺 I 带宽增加。无法对>1680 cm（-1）区域中增加的拉曼强度进行明确的归属，但它可能与吸附状态下蛋白质分子缔合所涉及的结构变化有关，这有助于解释在洗脱条件下 pH 值为 4 时吸附在色谱颗粒上的单克隆抗体的近 100%保留率。
