Related Experiment Video
Updated: May 13, 2026
Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
Published on: May 1, 2019
Assessing Fc glycan heterogeneity of therapeutic recombinant monoclonal antibodies using NP-HPLC
1Biologics Research, Janssen Research and Development, LLC, Radnor, PA, USA.
Abstract:
Recombinant monoclonal antibodies (rMAbs) are becoming major human therapeutics to treat life-threatening diseases such as cancer. These rMAbs are produced using either in vitro cell culture processes or transgenic technology in animals or plants. Glycans present in the Fc region can affect functions of rMAbs. These Fc glycans are heterogeneous and impact binding of rMAbs to Fc gamma receptors (FcγRs) and C1q protein. As a result Fc glycans affect antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of rMAbs. Thus understanding the glycan heterogeneity is necessary during the development of these rMAbs as human therapeutics. Because of their biological significance, understanding the glycan structure and their impact on the function of antibody molecules is also a regulatory requirement. Glycan mapping by NP-HPLC with fluorescence detection is a sensitive and reproducible method. Labeling of released glycans with anthranilic acid (AA) using reductive amination procedure improves sensitivity of detection. The NP-HPLC method resolves both neutral and sialylated glycans, thus enabling the user to obtain a broad heterogeneity profile of Fc glycans in a single run. Added advantage of the method is that the labeled glycans can be characterized using mass spectrometry and the method is also amenable for LC-MS analysis.
Insights
Understanding Fc glycan heterogeneity in recombinant monoclonal antibodies (rMAbs) is crucial for therapeutic efficacy. A sensitive NP-HPLC method with fluorescence detection aids in characterizing these glycans, impacting drug development.
Area of Science:
- Biopharmaceutical development
- Glycobiology
- Protein therapeutics
Background:
- Recombinant monoclonal antibodies (rMAbs) are vital therapeutics for diseases like cancer.
- Fc-region glycans significantly influence rMAb effector functions by affecting Fc gamma receptor (FcγR) and C1q binding.
- Fc glycan heterogeneity impacts antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
Purpose of the Study:
- To highlight the necessity of understanding Fc glycan heterogeneity for rMAb therapeutic development.
- To emphasize the regulatory importance of characterizing glycan structures and their functional impact.
- To present a sensitive and reproducible analytical method for Fc glycan profiling.
Main Methods:
- Glycan mapping using Normal Phase High-Performance Liquid Chromatography (NP-HPLC) with fluorescence detection.
- Labeling of released glycans with anthranilic acid (AA) via reductive amination to enhance detection sensitivity.
- Characterization of labeled glycans using mass spectrometry (MS) and suitability for Liquid Chromatography-Mass Spectrometry (LC-MS).
Main Results:
- The NP-HPLC method effectively resolves both neutral and sialylated glycans in a single run.
- This technique provides a comprehensive heterogeneity profile of Fc glycans.
- The method offers improved sensitivity for glycan detection and allows for further MS-based characterization.
Conclusions:
- Accurate Fc glycan profiling is essential for the development and regulatory approval of rMAb therapeutics.
- The described NP-HPLC method is a sensitive, reproducible, and versatile tool for Fc glycan analysis.
- This analytical approach supports the optimization of rMAb function and efficacy by understanding glycan impact.
