Kinetic model-aided process optimization via integrated in-situ FTIR and real-time oscillatory shear rheometry: A case study on impurity control and gelation mitigation for a solid phase peptide cleavage process

Solid phase peptide synthesis is a widely used technique for the production of medicinal peptides, particularly when using unnatural amino acids. In such processes, when the synthesis is complete and the designed peptide sequence is obtained, a subsequent peptide isolation technique is often employed using a trifluoroacetic acid (TFA)/dichloromethane (DCM) solution mixture to promote the cleavage of the peptide from the resin while retaining the protecting groups on the peptide (soft cleavage). The peptide is then isolated via distillation, precipitation/crystallization, and drying unit operations. Throughout these processes, the reaction conditions must be optimized to minimize peptide degradation in the acidic cleavage solution. Furthermore, any peptide network formation during cleavage can dramatically increase the solution viscosity which poses significant operating risks in manufacturing. In this study, a systematic model-aided kinetics and rheology analysis is performed to rapidly identify optimal reaction conditions using the minimum number of screening experiments. The reaction kinetics investigation was conducted with the aid of a mid-FTIR probe to track peptide and TFA solution concentrations during the reaction in real-time. A detailed kinetics model of peptide cleavage and impurity formation is proposed and the adsorption isotherm of TFA is also considered. To confirm that an acceptable solution viscosity can be maintained at the specified reaction conditions over the duration of the projected process time, in-situ oscillatory rheometry was conducted to monitor any viscosity and modulus increase in the soft-cleavage reaction medium. The novel combination of reaction kinetics modeling, impurity control techniques, and the development of a peptide network phase diagram provides a layered approach that can be utilized in the rapid rational design of any solid phase peptide isolation processes.