At-line Solutions for Upstream Bioprocessing - APPLICATION NOTEBOOK

Importance of Topic

Modern biotherapeutic development relies on rapid, reliable, and user-friendly at-line analytical solutions to monitor both process parameters and product quality attributes. Integrating advanced LC-MS with automated sample preparation and intuitive informatics bridges the gap between core analytical laboratories and bioprocess teams, accelerating decision-making in upstream cell culture and accelerating time to clinic.

Study Objectives and Overview

This collection of application notes from Waters demonstrates end-to-end at-line solutions for upstream bioprocessing. Key goals:

• Implement high-resolution LC-MS workflows for cell culture media nutrient and metabolite profiling
• Monitor intact monoclonal antibodies (mAbs) and subunit product quality attributes (glycans, modifications)
• Develop high-throughput automated sample preparation using the Andrew+ pipetting robot
• Provide walk-up interfaces and integrated informatics (waters_connect, OneLab) for non-MS experts

Methodology and Instrumentation

Advanced technologies combined in unified workflows:

• Liquid Chromatography–Mass Spectrometry: BioAccord™ LC-MS System with ACQUITY™ Premier columns and RDa/Tof detection for cell culture media and protein/subunit analysis
• Automation: Andrew+™ Pipetting Robot (Extraction+ and Heater/Shaker+ modules) for automated dilution, Protein A capture, FabRICATOR digestion, glycan labeling, buffer exchange, and cleanup
• Informatics: waters_connect™ and UNIFI™ platforms with dedicated applications (Intact Mass™, Peptide MAM, Glycan Screening) to automate data acquisition, processing, trending, and reporting
• Integration: Sample information exchange with Sartorius Ambr® high-throughput reactors via OneLab data interface

Results and Discussion

1. Cell Culture Media Profiling:

• Screened 200+ compounds in spent media using nine-minute LC-MS method
• Accurate monitoring of amino acids, vitamins, nucleotides, and metabolites with multivariate data analysis (MVDA)

2. Intact mAb Monitoring:

• Rapid 2.4-minute SEC-FLR method for direct titer and aggregate analysis in cell culture samples
• High sensitivity with 0.05–0.5 µL sample loads and extended column lifetimes

3. Subunit and Peptide-Level Attribute Analysis:

• High-throughput FabRICATOR digestion and LC-MS subunit screening for glycoform distribution and low-level PTMs
• Automated RapiZyme™ trypsin digestion and focused peptide mapping to quantify site-specific modifications (oxidation, deamidation, glycation)

4. Glycan Analysis:

• Automated GlycoWorks™ RapiFluor-MS protocol with added diafiltration for mAbs in any buffer or concentration
• Five-minute UPLC-MS method yields reproducible high- and low-abundance glycan profiles

5. Walk-Up Solutions:

• OneLab interface to trigger Andrew+ sample prep and BioAccord acquisition in a single walk-up workflow
• Bioprocess engineers can generate intact mass and media data at point of need without LC-MS expertise

Benefits and Practical Applications

• High throughput: 48-sample prep in 2–4 hours and sub-10-minute sample LC-MS runs
• Low sample consumption: µg-level loads suitable for microbioreactors
• Flexibility: workflows adaptable to any buffer or sample concentration
• Data quality: high sensitivity, reproducibility, and integrated reporting templates
• Empowerment: non-specialists can perform advanced LC-MS analytics on-site

Future Trends and Potential Applications

Continued evolution will focus on:

• Deeper multi-attribute monitoring combining intact, subunit, peptide, and glycan mapping in a single run
• Expanded on-board data analytics with AI-driven process insights via MVDA and predictive modeling
• Integration with other PAT tools (online UV, Raman, HPLC) for real-time bioreactor control
• Application to novel modalities: fusion proteins, cell and gene therapies, oligonucleotides

Conclusion

By combining robust LC-MS platforms, advanced sample preparation automation, and user-friendly informatics, Waters At-line Solutions for Upstream Bioprocessing enable rapid, reproducible, and accessible process and product attribute monitoring. These technologies empower bioprocess engineers to make data-driven decisions in real time, shortening development timelines and enhancing product quality.

References

1. Alelyunas YW et al. Cyt. J. Bioprocess Technol. 2021; 720007359EN
2. Shion H et al. Waters Application Note 720007547. 2022
3. Koza SM et al. Waters Application Note 720008042. 2023