Physicochemical Characterization of Coformulated Monoclonal Antibodies Using Agilent Biopharma Analytical Workflow Solutions

Significance of the Topic

Coformulating multiple antibodies into a single therapeutic agent offers the potential to target several disease pathways simultaneously and improve clinical outcomes. However, mixing mAbs increases the complexity of their physicochemical behavior and critical quality attributes (CQAs), demanding advanced analytical strategies to ensure safety, efficacy and stability.

Objectives and Study Overview

This study aims to evaluate an integrated analytical workflow for coformulated mAbs, focusing on charge variant profiling, aggregate quantitation and detailed peptide mapping for post-translational modifications (PTMs). Commercially available mAb1 and mAb2 served as model systems combined in a 1 to 1 ratio to assess method performance.

Methodology

An experimental design was developed encompassing three key techniques:

• Cation exchange chromatography with a pH gradient (pH 7 to 8.4) on an Agilent 1290 Infinity II bio LC quaternary pump and a Bio Mab NP5 ion-exchange column
• Size exclusion chromatography using the AdvanceBio SEC 300Å column on an Agilent 1290 Infinity II binary pump to quantify monomer and aggregates
• Tryptic peptide mapping with an AdvanceBio peptide mapping column coupled to Agilent 6545XT Q-TOF for sequence coverage and PTM analysis

Instrumental Setup

• Agilent 1290 Infinity II bio high-speed pump and flexible pump
• Multisampler and multicolumn thermostat from the 1290 Infinity II series
• Diode array detector for UV monitoring at 280 nm
• Agilent AdvanceBio SEC and Bio Mab NP5 columns
• AdvanceBio peptide mapping column on Agilent 6545XT LC Q-TOF with positive ESI
• OpenLab CDS and MassHunter BioConfirm software for data acquisition and analysis

Main Results and Discussion

• Charge Variant Analysis The pH-gradient CEX method resolved main, acidic and basic isoforms of each mAb and the coformulated mixture with retention time RSD below 0.17 % and area RSD below 10 %
• Aggregation Assessment SEC profiles showed monomer and aggregate peaks for individual mAbs and the coformulated sample. Although monomer separation was limited, the method provided reproducible quantitation of co-aggregates with area RSD below 4 %
• Peptide Mapping and PTMs Tryptic digests achieved greater than 95 % sequence coverage for both mAbs individually and in combination. Extracted ion chromatograms of antigen-binding region peptides confirmed identity, while oxidation, deamidation and lysine truncation levels were consistent across samples

Benefits and Practical Applications

This workflow delivers a comprehensive toolkit for quality control of coformulated mAbs. It enables simultaneous monitoring of charge heterogeneity, aggregate content and site-specific PTMs, streamlining stability studies and regulatory submissions.

Future Trends and Opportunities

• Expanding multi-attribute methods that integrate UV, MS and light scattering data
• Automation and high-throughput screening for large mAb libraries
• Application of artificial intelligence for pattern recognition in complex chromatograms and spectra
• Continuous real-time monitoring during bioprocessing to ensure consistent product quality

Conclusion

The Agilent biopharma analytical workflow effectively characterizes coformulated mAbs, offering high resolution and reproducibility for charge variants, aggregates and PTMs. These methods support robust CQA monitoring, facilitating the development of next-generation combination antibody therapies.

References

1. Larbouret C et al Cancers Basel 2021 13 4620 impact of antibody combinations on therapeutic efficacy
2. Mouchahoir T Schiel JE Anal Bioanal Chem 2018 410 2111 development of a peptide mapping protocol for the NISTmAb