(M1230-02-06) Application of icIEF-UV/MS for Rapid Proteoform Characterization and Charge Variant Analysis of a Novel Antibody-Drug Conjugate in Drug Discovery

Purpose: Protein therapeutics are known to produce various charge variants due to processes such as post-translational modifications. The analysis of these charge variants is critical as they may influence binding affinity and efficacy of the protein therapeutics. In the case of antibody-drug conjugates (ADCs), charge variation can also arise from differences in the drug-to-antibody ratio (DAR) or other chemical reactions on the linker or the drug, for example, ring opening events, these various sources of charge heterogeneity make it difficult to apply conventional charge variant analysis methods, particularly in the early drug discovery phase. In this study, we employed the Intabio ZT system, a microfluidic chip-based integrated icIEF-UV/MS system, to effectively analyze charge heterogeneity of a novel ADC. It combines imaged capillary isoelectric focusing (icIEF) separation with UV detection and high-resolution mass spectrometry (MS) technology, enabling simultaneous 2-dimentional analysis of charge-based separation and mass detection to accurately identify protein proteoforms, with minimal sample handling and a 30 min acquisition time.

Methods: The icIEF-UV/MS analysis was performed with Intabio ZT system form SCIEX. A master mix was prepared by mixing 3% of each Pharmalyte solution (pH 8-10.5 and 5-8) with 15 mM arginine and mixed with 400–800 µg/mL of desalted samples. Peptide markers with isoelectric points (pI) of 5.52 and 9.68 were added and used to estimate the pI values of the samples. The icIEF separation process was monitored in real-time using UV absorbance imaging with 280 nm wavelength. The focusing process lasted 6.5 minutes, during which the voltage increased stepwise: 1500 V for 1 minute, 3000 V for 1 minute, and 4500 V for 4.5 minutes. After focusing, peak mobilization was performed over 10 minutes using a voltage of 3000 V and an ESI tip voltage of 5500 V. Each charge variant was mobilized and introduced into the ZenoTOF 7600 system for high-sensitivity and high-resolution MS detection, providing data on mass shifts and isoform profiles. Finally, the data were analyzed using Biologics Explorer software, which provides a consolidated view of all the results.

Results: This study focused on the analyses of a monoclonal antibody (CNU001) and its ADC (CNU003). CNU003 is an ADC form of CNU001, containing a linker-drug GGFG-Deruxican attached to the light chain's mutated cysteine residue. When analyzing CNU001, the main peak revealed 13 distinct glycoforms. Among them, a series of glycoforms were observed with mass differences of 162 Da, indicating major glycosylation pairs with different numbers of terminal galactose. Moreover, the relative MS intensity of higher glycosylation pairs increased along with the acidic shift, suggesting that both neutral glycosylation and acidic glycation were present. This distinction could not be resolved using MS alone without sample deglycosylation treatment but was successfully differentiated using the Intabio ZT system in a single analysis. The basic variants were predominantly identified as C-terminal proline amidation, whereas the acidic variants included proteoforms with modifications such as sialic acid addition, deamidation, and mass shifts of 57 Da and 440 Da. In CNU003, three major charge variants on DAR2 were identified with a stepwise increase of +18 Da along with the acidic shift, a unique feature which was not observed in CNU001. These shifts were attributed to payload ring-opening events, which caused the profile to move toward a more acidic pI range. The stepwise +18 Da modifications occurred alongside proline amidation in the basic peak, while in the acidic peak, they were accompanied by a +440 Da shift.

Conclusion: In this study, we used the SCIEX Intabio ZT system to analyze charge heterogeneity in both CNU001 and CNU003. With significantly reduced time and sample preparation required, the system was able to provide an in-depth characterization of charge variants of both naked antibody (CNU001) and its ADC (CNU003). Notably, the study uncovered distinct glycoforms and modifications with different pI shifts, with CNU003 showing unique features such as +18 Da shifts attributed to payload ring-opening events.