Lectin-Based Fluorescent Comparison of Glycan Profile-FDA Validation to Expedite Approval of Biosimilars

Abstract

Glycan profile comparisons are one of the most tedious analytical exercises for establishing compliance with recombinant therapeutic protein batches. Based on its intensive research, the FDA has confirmed that lectin array binding with fluorescent monitoring is the fastest and most reliable method for profile comparisons. Using a database of over 150 biological products expressed in nine diverse mammalian cell systems, the FDA immobilized 74 lectins to study their binding using fluorescently labeled glycoproteins. The FDA identified nine distinct lectins from a custom-designed lectin microarray: rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHALs, which detect core fucose, terminal GlcNAc, terminal β-galactose, high mannose, α-2,3-linked sialic acids, α-2,6-linked sialic acids, bisecting GlcNAc, terminal α-galactose, and triantennary structures, respectively. This method can be used for screening and routine testing and to monitor batch-to-batch variability of therapeutic proteins, including establishing analytical similarity as a crucial part of biosimilar development.

Keywords: FDA; antibodies; biosimilars; fluorescent monitoring; lectins; microarray.

Figure 1

This illustration depicts the basic structure of an antibody, featuring two indistinguishable heavy chains (colored blue) and two indistinguishable light chains (colored brown) that combine to form a Y-shaped configuration. The varied domains of the Fab region account for the antibody’s exceptionally precise affinity for an epitope on the surface of an antigen. The Fc region of the antibody engages with different immune cells to facilitate immunological reactions. The addition of various carbohydrate molecules (shown by colored geometric shapes) to each heavy chain forms glycans, which have a significant impact on Fc-mediated processes such as antibody-dependent cell-mediated cytotoxicity and other immune effector mechanisms. Therefore, glycans are essential quality features to consider when evaluating antibody-based treatments.

Figure 2

An illustration of the technique devised by CDER researchers for efficiently analyzing glycosylation patterns in manufacturing monoclonal antibodies (mAbs). This method utilizes lectins, a group of proteins that selectively bind to different glycan structures in mAbs. CDER’s research has discovered nine lectins, each with a specific affinity for one of nine commonly occurring epitopes in mAb therapies. Subsequently, these lectins were fixed onto a glass chip. The tested glycosylated antibodies are marked with a fluorescent molecule called Cy3. By incubating them on the nine-lectin chip and using a fluorometer to measure the fluorescence intensity of each lectin-containing well, one can obtain quantitative data regarding the unique glycan epitopes on the antibody [26,36].