A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates

摘要: Antibody drug conjugates (ADCs) are complex therapeutic agents combining the specific targeting properties of antibodies and highly potent cytotoxic small molecule drugs to selectively eliminate tumor cells while limiting toxicity normal healthy tissues. One unique critical quality attribute ADCs is content unconjugated present from either incomplete conjugation or degradation ADC. In this work, size exclusion chromatography (SEC) was coupled with reversed-phase (RP) HPLC in an online 2-dimensional format for identification quantitation related impurities ADC samples directly without sample preparation. The SEC method 1st dimension not only separated intact ADC, but also provided information about variants (monomer, dimer, aggregates, etc.) peak trapped sent a RP-HPLC 2nd further separate quantify different sample. This SEC–RP 2D-LC demonstrated excellent precision (%RSD < 2.0), linearity (r2 = 0.9999), sensitivity (LOQ 0.05 μg/mL free sample) accuracy (95–105% recovery spiked samples). utilized study stability product at temperatures pHs. Both products aggregation conjugate were observed pathways investigated. offers powerful tool characterization provides valuable formulation development.