(T1330-06-32) Identification and Control Strategy of Diastereomer Impurities in API Produced via Biocatalytic Cascade

Purpose: The API is an investigational small molecule antiviral. Due to the need for novel antivirals, a 3-step, biocatalytic route was developed, allowing for significant reduction in step count and improved yield over traditional synthetic routes. The reduced route and enzymatic component presented various novel analytical challenges including two unknown and unspecified impurities routinely observed above specification ( >0.10 area%) in lab development samples. Their consistent presence necessitated urgent structural identification and control strategy implementation to facilitate large scale batch production.

Methods: Reverse phase UHPLC-DAD was employed for purity assay and spectroscopic analysis of impurities. LC-HRMS/MS analysis was used for structural elucidation of impurities. UHPLC-MS was implemented for quantification of diastereomer impurities with a sufficiently low LOQ ( < 75 ppm) for the control strategy.

Results: UHPLC-DAD chromatograms demonstrated that both impurities shared a unique characteristic of absorbing at 210 nm, but not 260 nm. This inability to absorb at 260 nm suggested the pi bond conjugation in the API’s six-member ring is broken. LC-HRMS/MS analysis found molecular weight and fragmentation pattern of both impurities were identical, signifying the impurities were a pair of diastereomers. Further testing confirmed the diastereomers arise from the over-addition of hydroxylamine sulfate during the API step. The diastereomer impurities were assessed using in silico tools as per ICH M7, and required a control strategy to limit them to < 75 ppm combined, until additional derisking can be performed. Leveraging process knowledge from the optimization of traditional chemistry routes, the team developed a control strategy by employing a refined isolation procedure that converts these diastereomer impurities back to the desired API form under controlled acidic conditions. Control strategy reduced presence of diastereomer impurities from ~8500 ppm to < 75 ppm at lab scale. HPLC-MS quantification was implemented in the API release to verify the impurities were reduced below the 75 ppm limit. In a confirmation of the robust control strategy, the new isolation process was successfully transferred to a third party CMO and three 30 kg batches were produced with the diastereomer pair maintained below the 75 ppm limit.

Conclusion: Structure of the two unknown peaks was determined through LC-HRMS/MS analysis. The diastereomers levels are being controlled as per ICH M7, with a control strategy comprising of a redesigned isolation procedure and implementation of LC-MS analysis capable of sufficient LOQ ( < 75 ppm). The control strategy was successfully transferred and deployed during large scale production of the API using the novel biocatalytic route.


LC Impurity Profile at Two Wavelengths (260 and 210 nm)


Representative LC-HRMS/MS data