The α-chymotrypsin-based cleavage reaction is necessary for manufacturing peptides using rDNA technology with tandem repeats. The current work showcases application of process analytical technology (PAT) tools for monitoring and control of this reaction, using recombinant Lethal Toxin Neutralizing Factor (rLTNF) as a case study. At-line Fourier Transform infrared spectroscopy (ATR-FTIR) combined with attenuated total internal reflectance sampling accessory was exploited to monitor the reaction. PLS spectral calibration models were created for real-time quantification of concentrations of rLTNF concatemer and urea in the reaction mixture. An end-to-end PAT monitoring and control strategy was developed to address potential deviations and ensure that targets for yield, purity, and impurity profile are met for each batch. The impact of various deviations of process parameters outside the operating space, such as deviations in the reaction buffer, concentration of concatemer in the IBs, enzyme loading relative to protein concentration, and reaction time with late quenching were investigated. Variation in impurity profile over time in the case of late reaction quenching was determined through HPLC and mass spectrometry. It has been demonstrated how process signatures from the PAT tools across various batches and campaigns can be analyzed to facilitate real-time process monitoring and control.

中文翻译：

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肽生产的过程分析技术的实现：以重组致死毒素中和因子串联体的裂解反应为例。

使用带有串联重复序列的rDNA技术生产肽时，基于α-胰凝乳蛋白酶的裂解反应是必需的。当前的工作展示了使用过程分析技术（PAT）工具监测和控制该反应的应用，以重组致死毒素中和因子（rLTNF）为例。在线傅里叶变换红外光谱（ATR-FTIR）与衰减的全内反射采样附件相结合来监测反应。创建了用于实时定量反应混合物中rLTNF串联体和尿素浓度的PLS光谱校准模型。开发了端到端的PAT监视和控制策略，以解决潜在的偏差，并确保每个批次都达到产量，纯度和杂质分布的目标。研究了操作空间外部各种工艺参数偏差的影响，例如反应缓冲液中的偏差，IBs中串联体的浓度，相对于蛋白质浓度的酶负载以及后期淬灭的反应时间。通过HPLC和质谱法确定在晚反应淬灭的情况下杂质分布随时间的变化。已经证明如何分析来自不同批次和活动的PAT工具的过程签名，以促进实时过程监视和控制。通过HPLC和质谱法确定在晚反应淬灭的情况下杂质分布随时间的变化。已经证明如何分析来自不同批次和活动的PAT工具的过程签名，以促进实时过程监视和控制。通过HPLC和质谱法确定在晚反应淬灭的情况下杂质分布随时间的变化。已经证明如何分析来自不同批次和活动的PAT工具的过程签名，以促进实时过程监视和控制。