Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Fritz-Haber-Weg 2, Karlsruhe, Germany.
Institute of Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen, Germany.
J Chromatogr A. 2018 Apr 27;1547:37-44. doi: 10.1016/j.chroma.2018.03.005. Epub 2018 Mar 5.
Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques.
傅里叶变换红外光谱（FTIR）是分析小分子和蛋白质二级结构的一种成熟的光谱方法。然而，FTIR 通常不适用于蛋白质色谱的在线监测。在三个案例研究中，研究了在线 FTIR 作为下游加工过程分析技术（PAT）的潜力，这些案例研究解决了当前应用的光谱 PAT 方法的局限性。第一项案例研究利用单克隆抗体（mAb）和溶菌酶的二级结构差异，通过偏最小二乘回归（PLS）选择性地定量这两种蛋白质，得到交叉验证均方根误差（RMSECV）分别为 2.42g/L 和 1.67g/L，相应的 Q 值分别为 0.92 和 0.99，表明在校准范围内具有稳健的模型。其次，监测了分离溶菌酶和聚乙二醇化溶菌酶的过程，对当前洗脱物种的聚乙二醇化程度进行了估计，RMSECV 分别为 2.35g/L 和 1.24g/L，Q 值分别为 0.96 和 0.94。最后，向溶菌酶的进料中添加了 Triton X-100，作为典型的与工艺相关的杂质。结果表明，该物种可以从 FTIR 3D 场中进行选择性定量，而无需 PLS 校准。总之，所提出的 PAT 工具具有作为监测蛋白质色谱的多功能选择的潜力。它可以通过减轻当前技术的局限性，有助于更完整地实施 PAT 计划。
