An Integrated Platform for Fully Automated, Continuous, & Real-Time Multi-Attributes Monitoring of Upstream Processes for Glycosylated Monoclonal Antibodies Production

The goal of this project is the development and validation of an integrated process analytical toolkit that enables fully autonomous and continuous monitoring of drug quality during biologics/biosimilars manufacturing.

Industry Need

The industry would benefit from a workflow that can rapidly monitor biological drug critical quality attributes (CQA) that can help enable advanced/continuous monoclonal antibody (mAb) biomanufacturing processes.

Approach

The team will develop and validate integrated online sampling process analytical technology (PAT) workflows to rapidly monitor biological drug critical quality attributes (CQA) to enable advanced/continuous monoclonal antibody (mAb) biomanufacturing processes.

The team will develop a PAT workflow called the ‘N-GLYcanyzer.’ N-GLYcanyzer integrates automated biologics sampling from upstream bioprocesses and mAb N-glycosylation analysis in real-time using integrated online sequential flow injection and liquid chromatography (LC).

The project will consist of:

Demonstration of downstream integration of N-GLYcanyzer with Agilent’s Infinity II Multisampler to monitor multiple CQA by means of InfinityLab Online LC Solutions in various bioreactor operational modes
Upstream integration of Raman flow cell prior to the N-GLYcanyzer to continuously monitor/predict mAb glycoforms and validated by the PAT workflow
Application of an integrated modeling framework to validate the PAT results and assess technoeconomic advantages for implementing real-time PAT with increased online sampling frequency
Independent validation by a federal stakeholder of the N-GLYcanyzer workflow in a series of round robin tests using CHO cell lines producing NISTmAb and model biosimilar Trastuzumab

Impacts

Development and application of a system based on flow injection technology for direct sampling, and integrated sample analysis using online LC/Raman based PAT to monitor multiple CQAs (like glycosylation, titer, charge states) in an autonomous manner.

Round robin validation of PAT workflow in collaboration with federal stakeholder (FDA CDER) that is currently deploying and testing the N-GLYcanyzer system

Robust integration of Agilent’s online multisampler LC system and Endress’s Raman flow cell technology with N-GLYcanyzer sampler unit to allow analysis of samples drawn directly from bioprocess unit operation for multiple CQA analysis

Use mechanistic modeling to validate predictions from N-GLYcanyzer in actual fed-batch and perfusion upstream process at varying PAT sampling frequency

Use technoeconomic analysis to explore pros/cons of online vs. offline PAT

Value Statement/Outcomes

This project validated an integrated online LC/Raman PAT platform that accelerates multi‑attribute CQA monitoring for both, resulting in time benefits across upstream mAb production.

Outputs/Deliverables

Development and validation of an UHPLC based PAT system to rapidly estimate N-glycans and amino acids at/on-line

Integration and validation of in-situ and at/on-line flow cell-based Raman PAT system for N-glycans and amino acids

Development of chemometric models using Raman in-situ/flow cell probes for real-time CQA prediction

Validated PAT systems robustness and analytical reproducibility through multiple fed-batch bioreactor campaigns

Development of mechanistic process models trained on PAT data for predictive control & design space evaluation

Technoeconomic analysis of real-time PAT implementation (like N-GLYcanyzer) using digital twin process models

Publications

Gyorgypal A, Fratz-Berilla E, Kohnhorst C, Powers DN, Chundawat SPS. Temporal Galactose-Manganese Feeding in Fed-Batch and Perfusion Bioreactors Modulates UDP-Galactose Pools for Enhanced mAb Glycosylation Homogeneity. Biotechnol Bioeng. 2025 Apr 18. https://doi.org/10.1002/bit.28999. Epub ahead of print. PMID: 40251805.

Gyorgypal, A., Chaturvedi, A., Chopda, V. et al. Evaluating the impact of media and feed combinations on CHO cell culture performance and monoclonal antibody (trastuzumab) production. Cytotechnology 77, 40 (2025). https://doi.org/10.1007/s10616-024-00690-7

Gyorgypal, A., Fratz-Berilla, E., Kohnhorst, C., Powers, D. N., & Chundawat, S. P., Temporal Effects of Galactose and Manganese Supplementation on Monoclonal Antibody N-Linked Glycosylation in Fed-Batch and Perfusion Bioreactor Operation.

Gyorgypal, A., Potter, O. G., Chaturvedi, A., Powers, D. N., & Chundawat, S. P. S. (2023). Automated instant labeling chemistry workflow for real-time monitoring of monoclonal antibody N-glycosylation. Reaction Chemistry & Engineering, 8(10), 2423-2434. https://doi.org/10.1039/D2RE00568A

Tiwold, E. K., Gyorgypal, A., & Chundawat, S. P. S. (2023). Recent Advances in Biologic Therapeutic N-Glycan Preparation Techniques and Analytical Methods for Facilitating Biomanufacturing Automation. Journal of Pharmaceutical Sciences, 112(6), 1485–1491. https://doi.org/10.1016/j.xphs.2023.01.012

Presentations

Chundawat, S. P. & Narvekar, A., An automated and fully integrated process analytical platform for combined analysis of N-glycans and amino acids to enable continuous manufacturing of glycosylated biologics, Bioprocessing Virtual Event Series 2024, Virtual, April 3, 2024.