Murphy Brian M, D'Antonio Jennifer, Manning Mark C, Al-Azzam Wasfi
BioPharm Analytical Sciences, BioPharmaceutical Development R&D, GlaxoSmithKline, King of Prussia, PA 19406, USA.
Curr Pharm Biotechnol. 2014;15(9):880-9. doi: 10.2174/1389201015666141012181609.
Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics.
证明治疗性蛋白质中作为高阶结构（HOS）一部分的二级结构组成的可比性是一项重大挑战。此前，我们表明二阶导数酰胺I傅里叶变换红外（FTIR）光谱的变异性足够小，以至于对于多种模型蛋白，可以观察到二级结构的显著差异。这些比较使用光谱重叠和光谱相关系数来量化光谱差异。然而，下游纯化过程、原料药和药品制剂中使用的许多辅料，如游离氨基酸和糖类，会干扰酰胺I区域的吸光度。在本研究中，酰胺II FTIR光谱分析被证明是一种替代方法，可用于替代使用酰胺I区域的光谱数据来分析蛋白质二级结构以评估其高阶结构。本研究提供了用于分析酰胺II FTIR光谱以证明蛋白质二级结构可比性的光谱重叠和光谱相关系数数学方法。光谱重叠和光谱相关系数结果表明，在不同条件下，各种模型蛋白的酰胺II和酰胺I FTIR光谱二阶导数的变化之间存在强相关性，这证明了使用酰胺II FTIR光谱来比较蛋白质二级结构的适用性。这些结果表明，酰胺II FTIR光谱二阶导数分析可用于监测和证明蛋白质治疗药物下游工艺和制剂开发过程中蛋白质二级结构的可比性。
