ANALYSIS OF MONOCLONAL-ANTIBODY AND IMMUNOCONJUGATE DIGESTS BY CAPILLARY ELECTROPHORESIS AND CAPILLARY LIQUID-CHROMATOGRAPHY

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using c apillary electrophoresis (CE) and capillary liquid chromatography (CLC ). A unique, highly sensitive and selective approach combined with bot h UV absorbance and laser-induced fluorescence (LIF) detection has bee n developed and applied to studies including enzymatic digests of anti body and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique c haracteristic of the anticancer drug DOX which yields native fluoresce nce. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches w ere developed based on the successful conjugation and analysis of a mo del peptide conjugate. Enzymatic digests of the monoclonal antibody BR 96 and its immunoconjugate were mapped by CE and CLC with on-line UV a nd LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-D OX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detecte d, which can be further characterized using other complementary analyt ical techniques.