BioRationality: FDA Simplifies Glycan Profiling of mAbs, a Significant Step in Reducing the Cost of Biosimilars

The most critical test of biosimilarity for recombinant therapeutic proteins derived mainly from Chinese hamster ovary cells, but others as well, is the comparison of post-translational modifications, of which the glycosylation is the most critical but also most variable and complex to test, adding substantial cost of batch release, and thus the cost of goods sold, as well as establishing biosimilarity.

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Glycosylation is crucial for antibody function, affecting stability, pharmacokinetics, effector functions like complement activation and antibody-dependent cellular cytotoxicity, and immunogenicity. For non-antibody biologics, glycosylation patterns vary widely depending on the specific biologic and the expression system used, influencing stability, solubility, and receptor binding affinity. Approximately 50% to 60% of all human proteins are glycosylated. Variance in glycosylation comes from the genetic sequences used to express the protein in cells, the conditions present during large-scale cell culture (such as nutrient levels, pH, oxygen availability, and cell density), and the purification methods employed. One challenge in this ongoing monitoring process is the complexity of current analytic methods that involve removing the glycan from the protein backbone and utilizing various separation techniques and mass spectrometry to identify the carbohydrate molecules based on their precise molecular masses.

To address the need for a simple, rapid, and high-throughput method for analyzing glycosylation patterns in antibody-based therapeutics, FDA’s Center for Drug Evaluation and Research researchers explored the use of lectins to detect glycan epitopes in therapeutic antibodies.^1,2 Lectins are a diverse class of naturally occurring or bioengineered proteins that share a crucial property with antibodies, i.e., an affinity for specific structures. In the case of lectins, these structures are typically found on the surface or terminus of glycans, which, borrowing from the term used in antibody-antigen interactions, are also referred to as epitopes.³

The FDA investigators first analyzed the data in applications submitted to the FDA’s Electronic Common Technical Document system for more than 150 FDA-approved therapeutic antibody products from December 1994 to May 2023. These antibodies were produced through diverse mammalian cell expression systems. They identified 9 universal glycan epitopes present across all therapeutic monoclonal antibodies (mAbs). Following this, they immobilized 74 different lectins on glass chips, each exhibiting distinct binding affinities to various glycan epitopes. These lectins were then incubated with fluorescently labeled mAbs, for which prior knowledge about their glycan structures existed. By measuring the binding of various fluorescently labeled glycoproteins, the investigators identified 9 different lectins, each selectively binding to one of the 9 common epitopes. They constructed a microarray of these nine lectins. They validated the selectivity of each one for a particular glycan by conducting binding experiments with commercial antibodies possessing well-documented glycosylation profiles and 2 non-glycosylated therapeutic proteins. Furthermore, the specific binding of each of the 9 lectins was confirmed by using enzymes (glycosidases) to remove carbohydrate groups from the glycans and assessing whether changes in binding were consistent with the removed epitopes. Additional experiments with an innovator antibody and its three biosimilars confirmed the ability of the array to detect different glycosylation patterns consistent with proprietary data about these products.

This FDA study introduced a custom-designed lectin microarray featuring 9 distinct lectins: rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHAL. These lectins were tailored to selectively bind to common N-glycan epitopes found in therapeutic immunoglobulin antibodies. The 9-lectin microarray identified provides a high-throughput platform for rapid glycan profiling, enabling comparative analysis of glycosylation patterns by utilizing intact glycoprotein samples. The practical utility of this microarray in assessing glycosylation across various manufacturing batches or between biosimilar and innovator products is a pivotal change in the development of new biologicals and biosimilars.⁴

While the lectin-based assays offer a more straightforward solution and can be performed in a high-throughput manner, these are relatively semi-quantitative at best, with exact glycan composition and linkage details not fully resolved.^5,6 In contrast, traditional methods such as mass spectrometry, liquid chromatography, capillary electrophoresis, and nuclear magnetic resonance provide detailed information on glycan composition, linkage, and modifications.⁷ However, the FDA made a pivotal decision based on the rational approach that the purpose of testing is to ensure consistency of the glycan profile; its absolute value is of lesser importance. In establishing biosimilarity, therefore, lectin-based assays provide a quick initial assessment and provide sufficient proof that the FDA is now accepting it as relevant to establishing biosimilarity.

The FDA conclusions significantly impact the understanding of characterization vs comparison. The key issue is maintaining the glycan profile, whatever it is, as found in the clinical lots. This is a significant concept concerning comparison as a measure of biosimilarity that developers hesitated to adopt. Still, with the full support of the FDA, considerable costs and time have shrunk to the best advantage of the biosimilar industry.

I could not be more thankful to the FDA for adopting rational science over traditional conservative approaches that have long led to a waste of efforts and resources; the biosimilar industry will benefit significantly.

References

1. Rapid glycan profiling with a nine-lectin microarray for therapeutic IgG1 monoclonal antibodies. FDA. Updated June 2, 2023. Accessed July 22, 2024. https://www.fda.gov/science-research/fda-science-forum/rapid-glycan-profiling-nine-lectin-microarray-therapeutic-igg1-monoclonal-antibodies

2. A novel method for rapid glycan profiling of therapeutic monoclonal antibodies. FDA. Updated June 18, 2023. Accessed July 22, 2024. https://www.fda.gov/drugs/regulatory-science-action/novel-method-rapid-glycan-profiling-therapeutic-monoclonal-antibodies#:~:text=The%20lectin%20microarray%20for%20glycan,of%20these%20increasingly%20indispensable%20therapeutics.2024

3. Luo S, Zhang B. Benchmark glycan profile of therapeutic monoclonal antibodies produced by mammalian cell expression systems. Pharma Res. 2024;41(1):29-37. doi:10.1007/s11095-023-03628-4

4. Luo S, Zhang B. A tailored lectin microarray for rapid glycan profiling of therapeutic monoclonal antibodies. MAbs. 2024;16(1):2304268. doi:10.1080/19420862.2024.2304268

5. Lis H, Sharon N. Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev. 1998;98(2):637-74. doi:10.1021/cr940413g

6. Chrispeels MJ, Raikhel NV. Lectins, lectin genes, and their role in plant defense. Plant Cell. 1991;3(1):1-9. doi:10.1105/tpc.3.1.1

7. Sharon N, Lis H. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology. 2004;14(11):53R-62R. doi:10.1093/glycob/cwh122