Physicochemical characterization and functionality comparison of Humira®(adalimumab), Remicade®(infliximab) and...

Area of Science:

• Biopharmaceutical analysis
• Immunology
• Protein therapeutics

Background:

• FDA-approved anti-tumor necrosis factor-alpha (TNFα) biopharmaceuticals are effective for autoimmune diseases.
• Patient outcomes vary among anti-TNFα products, suggesting product-specific differences.
• These differences may arise from structural variations, dosage, and administration routes.

Purpose of the Study:

• To investigate structural and functional differences among three full-length anti-TNFα monoclonal antibodies (mAbs): Humira®, Remicade®, and Simponi Aria®.
• To understand how these differences influence product efficacy.

Main Methods:

• Structural characterization involved quantifying N-glycans using mass spectrometry and fluorescence labeling.
• Functional studies assessed TNFα binding (ELISA), FcγRIIIa binding (AlphaLISA), and effector function (ADCC bioreporter assay).

Main Results:

• Remicade® exhibited the highest percentage of afucosylated glycans (15.5 ± 1.3%) and the most unique glycans (28).
• Humira® showed the fewest unique glycans (15) and a lower percentage of afucosylated, high-mannose glycans (11.4 ± 0.8%).
• Humira® demonstrated significantly higher binding affinity for TNFα (EC50: 1.9 ± 0.1 pM) and FcγRIIIa (IC50: 10.5 ± 1.1 nM), and was most potent in the ADCC assay (EC50: 0.55 ± 0.03 nM).

Conclusions:

• Significant structural and functional differences exist between anti-TNFα mAbs, even those with similar mechanisms of action.
• Humira® exhibits superior binding affinities and effector function compared to Remicade® and Simponi Aria® under the tested conditions.
• These product-specific variations are critical for understanding differential patient outcomes.