Improving the Quality of Antibody Drug Conjugates by Orthogonal Analytical Methods

Significance of the Topic

Antibody drug conjugates (ADCs) combine the targeting specificity of monoclonal antibodies with the potency of cytotoxic payloads. The controlled attachment of drugs via linkers to antibody residues has enabled a new class of biotherapeutics in oncology and beyond. High precision in measuring drug to antibody ratio (DAR), aggregate levels, and positional isomers is vital for safety, efficacy and regulatory approval.

Objectives and Study Overview

This study reviews the implementation of orthogonal analytical methods to characterize critical quality attributes of ADCs. It outlines strategies for monitoring intact DAR distributions, subunit analysis, aggregation profiles and heterogeneity arising from cysteine versus lysine conjugation.

Methodology

Orthogonal chromatographic and mass spectrometric techniques were employed to capture complementary information on ADC structure and purity. Hydrophobic interaction chromatography (HIC) under native conditions was used to resolve DAR species of cysteine linked ADCs. Reversed phase chromatography coupled to mass spectrometry (RP MS) provided subunit separation and positional isomer resolution for lysine linked ADCs. Size exclusion chromatography (SEC) evaluated aggregate and fragment levels. Two dimensional liquid chromatography with native mass spectrometry enabled intact mass and DAR determination.

Instrumental Setup

The analytical platform combined:

• Agilent AdvanceBio HIC columns with high pore size and bioinert surfaces for native DAR analysis
• Agilent PLRP S polymeric reversed phase columns for subunit and peptide mapping via RP MS
• Agilent AdvanceBio SEC columns of varying pore sizes for aggregate quantitation
• Quaternary pump LC systems to manage high salt gradients and organic modifiers
• Agilent Jet Stream electrospray sources and QTOF mass spectrometers for sensitive mass detection

Main Results and Discussion

HIC profiles of a model ADC demonstrated clear separation of DAR 0 to DAR 8 species with increasing retention times reflecting hydrophobic payload loading. RP MS analyses resolved heavy and light chain fragments, confirming conjugation sites and payload distribution. SEC chromatograms quantified monomer, dimer and higher order aggregates, revealing column selectivity differences. The 2D LC native MS workflow successfully integrated HIC, SEC and mass detection to deliver comprehensive intact mass profiles and DAR distributions in a single analysis.

Benefits and Practical Applications

The combined orthogonal approach enhances ADC characterization by offering:

• Improved accuracy in DAR determination and impurity profiling
• Reduced development timelines via early structure function insights
• Robust quality control workflows aligned with regulatory guidelines
• Flexibility to adapt methods based on conjugation chemistry and sample complexity

Future Trends and Applications

Emerging directions include the development of microfluidic and high throughput LC platforms, novel stationary phase chemistries tailored for biotherapeutics, and deeper integration with artificial intelligence for automated data analysis. Advances in native MS and multidimensional separations will further streamline ADC development and support next generation conjugate formats.

Conclusion

Orthogonal analytical techniques combining HIC, RP MS, SEC and native mass spectrometry provide a comprehensive toolkit for ADC quality assessment. Tailoring method selection to conjugation strategy and target attributes enables rapid, reliable characterization essential for successful biotherapeutic development.

References

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