(W1130-09-57) Re-Development of a Fit-for-Purpose Assay Degradation HPLC Method Using Open Lab Software

Purpose: During the development and validation for a phase 1 program, a fit-for-purpose HPLC Assay/Degradation method was developed for small molecule compound A. However, it was noted that the assay method has robustness challenges. Unknown peaks were identified above ICH reporting threshold which were later identified to be non-API related. Method optimization was warranted to assure correct stability assay and degradation level quantitation at each time point. ACD/Labs and fusion QbD HPLC column screening tools allowed us to evaluate the chromatographic conditions comprised of six stationary/mobile phase combinations. The mobile phases utilized ranged from Mass Spectrometry compatible to non-Mass Spectrometry compatible, and cover a wide range of pH, and organic solvent strengths. The selection of mobile phase had a significant impact on the baseline performance. Screening tools offered information on critical method parameters that allowed a selection of the optimized chromatographic conditions. Optimization of the gradient condition using these column screening tools was rapid and significantly reduced the cost, labor, and waste production.

Methods: Due to Unknown peaks identified above ICH reporting threshold which were later identified to be non-API related, method optimization of the Fit-For-Purpose HPLC method was warranted. The Automation of data acquisition utilized open lab software such as ACD\Labs and Fusion. The HPLC column screening tool allowed us to evaluate the chromatographic conditions comprised of six columns (Water Acquity BEH C18, Water Acquity BEH shield RP18 , Water Acquity BEH Phenyl-Hexyl , ACE C18-PFP , ACE Ultracore Super C18 , Waters HSS T3) with stationary/mobile phase combinations. The mobile phases utilized ranged from MS compatible to non-MS compatible, and cover a wide range of pH, and organic solvent strengths. A systematic approach was used to identified key parameters that have an impact on the assay and degradation results. This was used to understand the robustness of the methods.

Results: An Assay Degradation HPLC Method was developed for a Phase 1 clinical small molecule compound A. Preliminary risk assessments informed us on the challenges faced when optimizing mobile phase composition and changing wavelength. Increasing the detection wavelength helped to remove interfering ghost peaks, however, the peak response of low-level degradation impurity peaks is also reduced. Formic Acid, Trifluoroacetic Acid and Ammonium Acetate were screened as potential mobile phase A modifiers. These gave exceptionally clean baseline, however, their absorbance at low detection wavelengths caused issues with the response of low-level peaks. Ammonium Phosphate as a mobile phase A modifier generated good peak separation and a clean baseline, but there is a potential of salt precipitation when elevated levels of Acetonitrile are reached. In the end, Phosphoric Acid will be maintained as the modifier and high-quality grade solvent will be used. With the use of the LC column screen software, the X-Bridge C18 column was chosen as the optimal column and an appropriate gradient was developed. A temperature of 35℃ was chosen as the appropriate temperature to obtain both high-resolution between the critical peaks and reduce peak tailing.

Conclusion: The use of LC screening software enabled selection of chromatographic conditions to improve peak resolution and prevent high noise levels. The method proved to be simple, accurate, precise, specific, and selective. The LC screening process optimized method is recommended for GMP analysis of clinical compounds degradation products for clinical stability samples. The LC screening software helped to eliminate the need to manually optimize the gradient and reduce the number of screening runs.

Figure 1. (A) Representative noise baseline with unknown peaks in Fit-For-Purpose Method

Table 1. Summary of Columns Evaluated

Table 2. Optimized Column Conditions