Development of a Size Exclusion Chromatography Cartridge-Based Analytical Method for Determination of Free Drug in Nano-liposomal Oncology Drug Formulations.

Abstract

A reliable and stability-indicating size exclusion chromatography (SEC) cartridge-based free drug testing method was developed for active loading nanoliposome formulations through a systematic development approach. SEC spin cartridge columns (7K MWCO, 2-mL) were used for developing liposome free drug testing procedure. SEC cartridge column retention capacity for Mirati drug was determined (445 µg per cartridge). SEC testing conditions were studied to achieve a good separation between drug-loaded liposomes and free drug, which include cartridge conditioning, buffer wash steps for liposomal drug elution, organic media wash steps for free drug elution, and sample size effect. Qualification of this newly developed SEC cartridge method has demonstrated its specificity / selectivity without interference, excellent detection linearity (correlation coefficient (R) > 0.999) over a study concentration range (1.0 to 46.9 µg/mL), sufficient LOQ (0.52 µg/mL equivalent to 1.7% of free drug in a liposome formulation at 2.5 mg/mL), acceptable accuracy / recovery of 81 to 89% for free drug in spiked samples at 4.5%, 9%, 18% levels (sample loading by a regular pipet) and 88 to 97% at 10% spike level (sample loading by a positive displacement pipet), good method precision (RSD (n=6) of not more than 4%) for free drug determination, respectively), and 3-day solution stability for both standard and sample solutions (2-8ºC). In comparative sample testing, SEC free drug test results were in a good agreement with solid phase extraction (SPE) test results for active loading formulations. New method’s stability-indicating ability has been proved through monitoring free drug in a discovery-stage liposome sample stored at -20ºC and -80ºC.