The global pharmaceutical market size reached approximately 262 trillion yen in 2024. Among these, biopharmaceuticals have shown remarkable growth, reaching 38 trillion yen in the same year and projected to continue growing at a rate exceeding 10%. Furthermore, biopharmaceuticals accounted for more than 50% of the top 100 pharmaceuticals in terms of global sales in 2019.
In anticipation of this situation, in 2011, FDA established a special team on therapeutic protein quality control, led by Dr. Baolin Zhang. The project focused on glycosylation of therapeutic antibodies and its impact on product safety and biological activity, particularly in relation to the evaluation of biosimilar products. The ultimate goal was to evaluate existing analytical tools worldwide and identify or develop analytical tools that could be used to better assess the quality of therapeutic antibodies. GlycoTechnica, the predecessor of Mx, delivered GlycoStation, a glycan profiling technology platform using lectin microarrays (LecChips), to FDA in 2013, and has been working with FDA for over 10 years to evaluate and develop lectin microarrays suitable for the analysis of therapeutic antibodies.
In 2016, FDA published two papers on the evaluation results of therapeutic antibodies using GSR1200 and LecChip Ver1.0.
Glycan analysis of therapeutic glycoproteins
The use of lectin microarray for assessing glycosylation of therapeutic proteins
In these papers, FDA explained the advantages of lectin microarrays for the evaluation of therapeutic antibodies as follows:
“Lectin microarrays enable direct measurement of the glycan profile of a protein, without the need for enzymatic digestion to remove glycans from the protein backbone. This type of platform technology for glycan analysis is unique in that it increases the likelihood of complete coverage of glycan variants on glycoproteins. Using a commercially available lectin microarray (LecChip Ver. 1.0), we were able to determine the glycan profiles of a series of therapeutic antibodies. In particular, lectin microarrays are highly sensitive to changes in terminal glycan epitope structures, i.e., galactosylation and sialylation, and lectin microarrays can distinguish between glycan isomers containing different sialic acid linkages effectively. Our data demonstrate the benefit of lectin microarrays for screening the glycan modification patterns of protein samples.”
Thus, while demonstrating the great potential of lectin microarrays for glycan analysis of biopharmaceuticals, FDA set the following goals for their ultimate implementation:
“The commercially available lectin microarray used in this study was not designed specifically for the evaluation of therapeutic antibodies. In principle, evaluation performance could be further improved by using lectins with enhanced selectivity and binding affinity for different glycan structures. To analyze specific glycoproteins, lectin microarrays can also be customized to include lectins related to the glycan structures that may be present in the test sample. By optimizing the lectin microarray in this way, this glycan analysis platform (GlycoStation) could potentially be employed as a useful tool for high-throughput screening of the glycan profiles of biopharmaceuticals.”
Based on this conclusion, FDA proceeded with the development of a custom lectin microarray for the evaluation of therapeutic antibodies using natural and recombinant lectins, totally 83 species, that GlycoTechnica was using at the time. The results were published in the following two papers in 2024.
Benchmark Glycan Profile of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems
A tailored lectin microarray for rapid glycan profiling of therapeutic monoclonal antibodies
The lectins used in this research and development were as follows:
LecChip Ver1.0
LecChip Ver2.0
FDA comprehensively analyzed the glycan modification structures of 157 approved therapeutic antibodies and discovered and identified nine commonly existing glycan epitopes: core fucose, terminal N-acetylglucosamine, terminal β-galactose, high mannose, terminal α2,3-linked N-acetylneuraminic acid, terminal α2,6-linked N-glycolylneuraminic acid, bisecting N-acetylglucosamine, terminal α-galactose, and triantennary structure. FDA carefully selected nine different lectins that selectively bind to these N-glycan epitopes and developed a custom-designed lectin array (IgG1-mAb-LecChip) for therapeutic antibodies evaluation in collaboration with GlycoTechnica. The nine lectins are rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHAL. This lectin array can be used directly on antibody drug samples without cleaving glycans, providing a high-throughput platform for rapid glycan profiling and enabling highly accurate comparative analysis of glycan modification patterns.
“FDA concluded that this lectin array is highly practical for assessing differences in glycan modification between various manufacturing batches or between biosimilars and innovator drugs.” IgG1-mAb-LecChip has 14 wells, each containing nine lectins spotted in triplicate. Simultaneous comparative glycan profiling of 14 different samples is possible.
