Multiple Critical Quality Attributes Assessment of mAbs for Process Control

Significance of the Topic

Monoclonal antibodies (mAbs) are among the most critical biopharmaceuticals, where subtle changes during production or purification can affect safety and efficacy. Continuous monitoring of critical quality attributes (CQAs) such as titer, aggregates, and charge variants is essential for robust process control, regulatory compliance, and consistent product performance.

Objectives and Study Overview

This application note describes an automated heart-cutting two-dimensional liquid chromatography (2D-LC) workflow using Agilent InfinityLab Online LC Monitoring Software. It integrates Protein A affinity chromatography for titer determination in the first dimension with size exclusion chromatography (SEC) or ion-exchange chromatography (IEC) for aggregates and charge variants in the second dimension, enabling real-time multi-attribute monitoring of mAbs.

Methodology

• Online sampling: 10 µL direct injections at 100 µL/min from a model rituximab solution to simulate bioreactor feed.
• First dimension: Protein A column captures mAb; DAD quantifies titer across a 0.2–10 g/L calibration range.
• Heart-cutting (0.75–0.95 min) transfers eluted mAb into a 180 µL loop for the second dimension.
• Second dimension: SEC or IEC separation via column switching valve; DAD detects aggregates or charge variants.
• Automated schedule: 12 blanks and analytical runs over ~34 hours to assess reproducibility.

Used Instrumentation

• Agilent 1290 Infinity II 2D-LC System with Online LC Monitoring Software
• Agilent 1260 Infinity II Online Sample Manager with external valve
• Diode array detectors (DAD) for both dimensions
• Protein A affinity, AdvanceBio SEC 300 Å, and Bio MAb NP5 IEC columns
• OpenLab CDS, 2D-LC Software, and Online LC Monitoring Software

Results and Discussion

Titer measurements matched the 7 g/L target: 7.13 g/L (1.15% RSD) with SEC and 7.17 g/L (0.94% RSD) with IEC. SEC profiling showed ~97.7% monomer, 0.16% high-molecular-weight aggregates, and 2.13% low-molecular-weight fragments. IEC analysis revealed ~83.9% main charge variant with acidic and basic variants. Trending plots in the software provided rapid visual QC across multiple CQAs.

Benefits and Practical Applications

• Consolidates multiple CQA assessments into a single automated workflow, reducing analysis time and sample handling.
• Supports near real-time process monitoring for upstream and downstream bioprocessing, enabling proactive control.
• Facilitates intact multi-attribute method (MAM) development without extensive sample prep.

Future Trends and Opportunities

Combining 2D-LC with advanced detectors (e.g., MS) can expand CQA coverage. Further integration of aseptic sampling devices will strengthen process analytical technology (PAT) deployments. Incorporating digital twins and AI-driven data analysis may enable predictive control of complex bioprocesses.

Conclusion

The Agilent InfinityLab Online LC Monitoring Software and heart-cutting 2D-LC workflow offer a powerful solution for simultaneous mAb titer, aggregate, and charge variant analysis. This approach streamlines process control and enhances data-driven decision-making in biopharmaceutical manufacturing.

References

1. Bonnington L.; Naegele E.; Kutscher D. Online LC Monitoring of Downstream Processing in the Production of Therapeutic mAbs. Agilent application note 5994-5186EN, 2022.
2. Chemmalil L.; Naegele E.; Kutscher D. An Automated Sampling System for Online Monitoring as a Process Analytical Technology (PAT) Tool. Agilent application note 5994-3716EN, 2021.
3. Sandra K.; et al. Determination of Multiple Attributes of Monoclonal Antibodies. Agilent application note 5994-3521EN, 2021.
4. Agilent InfinityLab 2D-LC ProtA-SEC Kit and Service. Agilent data sheet 5994-3630EN, 2021.