Alpha 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.