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FIA-Based Process Monitoring in Simulated Bioreactor for Monoclonal Antibody Production

Fernandez Alcantara, Leonardo Renzo LU (2025) KBTM01 20242
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
Research on monoclonal antibody (mAb) has considerably gained importance during the last decades, not only for cancer therapy but also for its importance in variety of fields. Unfortunately, the development phase can take several years before obtaining FDA approval, hence alternative approaches that could potentially shorten this timeline are critically needed. For instance, flow-injection analysis (FIA) delivers reproducible samples under controlled flow conditions without the need for manual pipetting or other interventions, in an online measurement manner.
In this project, we present a novel approach for sampling mAbs using FIA coupled with automation, where enzymes and actuators are integrated into the process to provide an almost... (More)
Research on monoclonal antibody (mAb) has considerably gained importance during the last decades, not only for cancer therapy but also for its importance in variety of fields. Unfortunately, the development phase can take several years before obtaining FDA approval, hence alternative approaches that could potentially shorten this timeline are critically needed. For instance, flow-injection analysis (FIA) delivers reproducible samples under controlled flow conditions without the need for manual pipetting or other interventions, in an online measurement manner.
In this project, we present a novel approach for sampling mAbs using FIA coupled with automation, where enzymes and actuators are integrated into the process to provide an almost real-time response, controlled by a custom software and tailor-made tubing system. By analysing and characterising the mAb subunits (i.e. middle-down analysis) and using Liquid Chromatography - Mass Spectrometry (LC-MS), we detect the glycan structures and post-translational modifications (PTMs) present in Trastuzumab. These results are the preliminary attempts to develop a closed-loop control system, where AI-driven tools could provide immediate feedback and be used to adjust upstream process parameters if the desired PTMs or glycan structures fail to develop during mAb cultivation. (Less)
Popular Abstract
Monoclonal antibody (mAb) therapy emerged as an alternative and complement to chemotherapy, aiming to reduce side effects and toxicity. Each mAb has a specific glycosylation pattern and post-translational modifications (PTMs), and detecting changes in these structures is crucial. An incorrect or unexpected modification can impact the mAb function, potentially impairing its ability to target cancer cells, preventing the intended immune response.
This project combines middle-down analysis of mAb using Liquid Chromatography -Mass Spectrometry (LC-MS) with flow-injection analysis (FIA), a technique that transports reagents through a network of tubing, enabling fast and reproducible detection of analytes under controlled flow conditions. The... (More)
Monoclonal antibody (mAb) therapy emerged as an alternative and complement to chemotherapy, aiming to reduce side effects and toxicity. Each mAb has a specific glycosylation pattern and post-translational modifications (PTMs), and detecting changes in these structures is crucial. An incorrect or unexpected modification can impact the mAb function, potentially impairing its ability to target cancer cells, preventing the intended immune response.
This project combines middle-down analysis of mAb using Liquid Chromatography -Mass Spectrometry (LC-MS) with flow-injection analysis (FIA), a technique that transports reagents through a network of tubing, enabling fast and reproducible detection of analytes under controlled flow conditions. The main attraction of this method lies in its inherent compatibility with automation, where a software controls the assay with pumps and valves, eliminating the need for manual pipetting and reagent handling.
This project proposed an online workflow where mAb samples were exposed to first, an IdeS enzyme, and then subsequent reduction steps to break disulfide bridges to analyse the Fc, Fd and Lc generated subunits and their respective glycan structures on LC-MS. The results demonstrate the ability of the system to detect any possible change and provide valuable insights mainly in the early-stage R&D by enabling rapid and frequent samples, which is crucial for real-time process monitoring, minimizing sample loss and contamination, while enabling near real-time feedback on the final product profile.
The ultimate future application of this project is to integrate mAb cultivation into a closed-loop control system, where after processing LC-MS results, immediate feedback can be used to adjust upstream process parameters such as pH, temperature, residence time, agitation, pH or other upstream parameters. (Less)
Please use this url to cite or link to this publication:
author
Fernandez Alcantara, Leonardo Renzo LU
supervisor
organization
course
KBTM01 20242
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Monoclonal antibody, Flow-injection analysis, Middle-down analysis, Automation, Liquid Chromatography - Mass Spectrometry, Biotechnology
language
English
id
9188201
date added to LUP
2025-05-09 10:19:19
date last changed
2025-05-09 10:19:19
@misc{9188201,
  abstract     = {{Research on monoclonal antibody (mAb) has considerably gained importance during the last decades, not only for cancer therapy but also for its importance in variety of fields. Unfortunately, the development phase can take several years before obtaining FDA approval, hence alternative approaches that could potentially shorten this timeline are critically needed. For instance, flow-injection analysis (FIA) delivers reproducible samples under controlled flow conditions without the need for manual pipetting or other interventions, in an online measurement manner. 
In this project, we present a novel approach for sampling mAbs using FIA coupled with automation, where enzymes and actuators are integrated into the process to provide an almost real-time response, controlled by a custom software and tailor-made tubing system. By analysing and characterising the mAb subunits (i.e. middle-down analysis) and using Liquid Chromatography - Mass Spectrometry (LC-MS), we detect the glycan structures and post-translational modifications (PTMs) present in Trastuzumab. These results are the preliminary attempts to develop a closed-loop control system, where AI-driven tools could provide immediate feedback and be used to adjust upstream process parameters if the desired PTMs or glycan structures fail to develop during mAb cultivation.}},
  author       = {{Fernandez Alcantara, Leonardo Renzo}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{FIA-Based Process Monitoring in Simulated Bioreactor for Monoclonal Antibody Production}},
  year         = {{2025}},
}