Analytical quality by design-based development and validation of a robust impurity analysis method for albumin-bound...

Analytical Quality by Design (AQbD) was applied to develop a robust impurity (IMP) analysis method for albumin-bound paclitaxel nanoparticles (Nab-PTX). Preliminary studies revealed that the USP method could induce epimerization, increasing IMP VI (7-epi-paclitaxel) formation. Risk assessment (Ishikawa/FMEA) identified four critical method parameters: flow rate, acetonitrile ratio (ACN), column temperature, and formic acid (FA) content. A 27-run Box-Behnken design (BBD) and response surface modeling (R² = 0.8688-0.9950) revealed complex nonlinear relationships. Specifically, FA content suppressed the dissociation of carboxylic acid groups in early-eluting impurities, significantly improving peak symmetry. To overcome standard software limits, a multidimensional Method Operable Design Region (MODR) was constructed using Monte Carlo simulation (n = 50,000) with a custom Python-based script. Optimal conditions (1.1 mL/min, 40.5% ACN, 25 °C, 0.025% FA) were identified via desirability function (D=0.9589). Experimental verification showed high predictive accuracy (Bias -1.4% to +1.0%). The method was validated per ICH Q2(R1) guidelines, showing excellent linearity (R²≥0.9985), accuracy (87.8-106.2%), and precision (RSD≤0.59%). Notably, acidified sample preparation reduced method-induced IMP VI formation by > 75%, preventing false-positives. This study provides a systematic AQbD framework with computational integration for protein-bound nanomedicines, ensuring superior resolution and 33% faster analysis than compendial methods.