Two-In-One Bioprocess Analytics - Combining mAb titer determination and spent media analysis by internal valve switching using the Agilent 1260 Infinity II Bio-Inert LC and InfinityLab LC/MSD iQ

Importance of the Topic

Process analytical technology (PAT) enables real-time monitoring of critical process parameters and quality attributes during biopharmaceutical manufacturing, ensuring optimal yields and product quality. Integration of multiple analyses on a single platform reduces footprint, cost, and manual operations while supporting Quality by Design principles.

Objectives and Overview

This study demonstrates a two-in-one bioprocess analytics workflow combining monoclonal antibody (mAb) titer determination and spent media analysis on a single Agilent 1260 Infinity II Bio-Inert LC system with automated valve switching and an InfinityLab LC/MSD iQ detector. The goal was to streamline at-line monitoring of cell culture nutrients and product concentration without manual reconfiguration.

Methodology

Spent media components were separated by hydrophilic interaction liquid chromatography (HILIC) using a Poroshell 120 HILIC-OH5 column and 30 mM ammonium formate buffer (pH 3) containing 5% 2-propanol. Mass detection in selected ion monitoring mode provided sensitive quantification of 16 polar analytes. mAb titers were measured by protein A affinity chromatography on a Bio-Monolith protein A column with phosphate buffer (pH 7.4) and acetic acid elution (pH 2.6) monitored at 280 nm.

Used Instrumentation

• Agilent 1260 Infinity II Bio-Inert pump, multisampler, and multicolumn thermostat with 4-position/10-port valve
• Agilent InfinityLab LC/MSD iQ for HILIC analysis
• Agilent 1260 Infinity II diode array detector for protein A assay
• Agilent OpenLab CDS version 2.4 software for automated method control and FDA 21 CFR Part 11 compliance

Main Results and Discussion

HILIC-MS analysis achieved linear calibration curves (R2 > 0.99) and detection limits below 1 pmol on-column for most compounds. Dynamic profiling of CHO cell cultures illustrated rapid consumption of glucose and glutamine and accumulation of lactic acid. Protein A chromatography revealed a peak mAb titer at day 6 followed by a decline, likely due to degradation. Automated valve switching enabled seamless alternation between HILIC and protein A methods without manual solvent or column changes. The Peak Explorer feature in OpenLab provided intuitive visualization of mAb production dynamics.

Benefits and Practical Applications

Combining HILIC and protein A assays on a single LC platform reduces hands-on time, instrument footprint, and method complexity. This approach supports at-line quality monitoring and process control in biopharmaceutical development and manufacturing under regulatory compliance.

Future Trends and Applications

Emerging trends include integration of real-time PAT with continuous bioprocessing, extension to additional analytes such as metabolites and host-cell proteins, miniaturized flow paths for higher throughput, and AI-driven data analytics for predictive process adjustments.

Conclusion

A two-in-one LC workflow offers a robust, automated solution for simultaneous nutrient profiling and mAb quantification, enhancing process understanding and enabling timely interventions within a Quality by Design framework.

References

• Tripathi N.K.; Shrivastava A. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Frontiers in Bioengineering and Biotechnology 2019.
• ICH Harmonised Tripartite Guideline Q8(R2): Pharmaceutical Development. 2009.
• Feith A. et al. HILIC-Enabled 13C Metabolomics Strategies: Comparing Quantitative Precision and Spectral Accuracy of QTOF High- and QQQ Low-Resolution Mass Spectrometry. Metabolites 2019.