Multilevel Characterization of a Chemoenzymatic Conjugated ADC by icIEF-UV/MS and RP-HPLC-MS EAD Fragmentation...

Area of Science:

• Bioconjugation Chemistry
• Analytical Chemistry
• Pharmacology

Background:

• Antibody-drug conjugates (ADCs) are crucial therapeutics combining targeted delivery with potent cytotoxic payloads.
• Developing homogeneous ADCs with controlled drug-to-antibody ratios (DAR) is essential for efficacy and safety.
• Enzyme-mediated conjugation offers a promising route for site-specific modification of antibodies.

Purpose of the Study:

• To synthesize monomethyl auristatin E (MMAE) payload conjugated Trastuzumab (TRA) using an enzyme-mediated glycan-remodelling reaction.
• To comprehensively characterize the resulting TRA-MMAE antibody-drug conjugates (ADCs) using multilevel analytical techniques.
• To establish a streamlined workflow for producing well-characterized and homogeneous ADCs.

Main Methods:

• Chemoenzymatic synthesis of TRA-MMAE conjugates.
• Microfluidic chip-based ultraviolet imaged channel isoelectric focusing mass spectrometry (icIEF-UV/MS) for intact proteoform analysis.
• Reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping with electron-activated dissociation (EAD) fragmentation.
• Determination of drug-to-antibody ratio (DAR) and site-specific conjugation analysis.

Main Results:

• Successful synthesis of TRA-MMAE ADCs with a high drug-to-antibody ratio (DAR 2) was achieved.
• icIEF-UV/MS enabled separation, quantitation, and identification of intact ADC proteoforms, revealing mass shifts indicative of conjugation.
• RP-HPLC peptide mapping with EAD fragmentation corroborated intact mass findings, localized posttranslational modifications, and validated site-specific MMAE conjugation to the Asn-300 glycan.
• The chemoenzymatic reaction demonstrated high yield and homogeneity of the TRA-MMAE ADC product.

Conclusions:

• The combined icIEF-UV/MS and RP-HPLC peptide mapping with EAD fragmentation provides a robust analytical strategy for ADC characterization.
• The evaluated chemoenzymatic conjugation reaction is effective for producing homogeneous TRA-MMAE ADCs with a defined DAR.
• This streamlined workflow facilitates the production and characterization of well-defined ADCs for preclinical evaluation and scale-up.