COMBINING INTACT GLYCOPROFILING AND CELL CULTURE MEDIA ANALYTICS FOR IN-PROCESS MONITORING OF AN AUTOMATED HIGH-THROUGHPUT MULTI-PARALLEL BIOREACTOR SYSTEM

Significance of the Topic

Rapid collection of process parameters and product attributes in bioprocessing is crucial for optimizing yield and ensuring quality.
Integrating intact glycoprofiling with media metabolite analysis allows real-time monitoring of critical quality attributes and nutrient flux within high-throughput bioreactor systems.

Objectives and Study Overview

The study demonstrates an automated workflow combining intact mAb glycosylation profiling and comprehensive spent media analytics for in-process control of a CHO cell–based monoclonal antibody production process within a high-throughput Ambr® 250 multi-parallel bioreactor platform.
Key aims:

• Assess direct intact protein analysis without affinity preparation.
• Quantify spent media nutrients and metabolites via direct dilution.
• Integrate LC-MS data into reactor control software for real-time feedback.

Methodology and Instrumentation

Samples were collected at days 4, 6, 8, 10, and 12 from five parallel bioreactors.
Post-collection, cultures were centrifuged, filtered (0.2 µm), then:

• Diluted 1:20 (V/V) for intact mAb analysis by LC-TOF MS (mass range 500–7000 m/z).
• Diluted 1:400 (V/V) for nutrient and metabolite profiling without derivatization.

Data acquisition used waters_connect™ and processed via Databridge for seamless transfer to Ambr® Control software.

Used Instrumentation

• Ambr® 250 High-Throughput 12 Station Bioreactor System (Sartorius Stedim).
• Andrew+™ pipetting robot (Waters Corporation).
• BioAccord™ LC-MS System (Waters Corporation).
• w waters_connect™ Informatics System with Databridge interface.

Key Results and Discussion

• Intact protein analysis revealed clear TIC signals with distinct light chain, intact mAb, and matrix peaks.
• Glycosylation profiles (% modifications) were tracked over time, showing consistent trends across bioreactors.
• Media analysis detected ~100 nutrients and metabolites; concentration changes were plotted per reactor and time point.
• Real-time integration of glycan and metabolite data into Ambr® software enabled direct process monitoring and rapid decision-making.

Benefits and Practical Applications

• Automated sample preparation streamlines throughput and reduces manual errors.
• Direct LC-MS analysis minimizes sample prep and accelerates data turnaround.
• Integrated data visualization supports real-time process control and optimization.
• High-throughput capability suits early-stage process development and scale-up studies.

Future Trends and Opportunities

Advancements may include:

• Expanding analyte panels for broader metabolomic coverage.
• Integration of AI-driven analytics for predictive process control.
• Enhanced connectivity with digital biomanufacturing platforms.
• Scale-up to commercial manufacturing with automated feedback loops.

Conclusion

The combined intact glycoprofiling and media analytics workflow delivers rapid, high-quality LC-MS data for in-process monitoring.
This approach enables improved bioprocess control, supports robust product quality assurance, and streamlines decision-making in high-throughput development environments.

Reference

• INTACT Mass™ - waters_connect™ Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics, 720007547.
• Monitoring Nutrients and Metabolites in Spent Cell Culture Media Using the BioAccord LC-MS System with ACQUITY Premier, 720007359.