Abstract
FDA-approved anti-TNFα biopharmaceuticals are successful in treating a range of autoimmune diseases. However, not all anti-TNFα products are identical in their patient outcomes, suggesting that there may be product-specific differences stemming from protein structural differences, doses and routes of administration. In this work, we focus only on structural and functional differences across three full-length anti-TNFα mAbs (Humira®, Remicade®, and Simponi Aria®) to better understand the implications of such differences on the products' efficacy. For structural characterization, we quantified N-glycans using mass spectrometry and fluorescence labeling. From these studies, we observed that Remicade® had the highest percent of afucosylated glycans (15.5 ± 1.3 %) and the largest number of unique glycans, 28. While Humira® had the fewest unique glycans, 15, and 11.4 ± 0.8 % of afucosylated, high-mannose glycans. For the functional studies we tested TNFα binding via ELISA, FcγRIIIa binding via AlphaLISA and effector function using an ADCC bioreporter assay. Humira® had a significantly lower EC50 (1.9 ± 0.1 pM) for ELISA and IC50 (10.5 ± 1.1 nM) for AlphaLISA, suggesting that Humira® has higher TNFα and FcγRIIIa binding affinity than Remicade® and Simponi Aria®. Humira® was also the most potent in the bioreporter assay with an EC50 value of 0.55 ± 0.03 nM compared to Remicade® (0.64 ± 0.04 nM) and Simponi Aria® (0.67 ± 0.03 nM). This comparison is significant as it highlights functional differences between mAbs with shared mechanisms of action when examined in a single laboratory and under one set of conditions.
Keywords:
Binding affinity; Bioanalytical comparison; Crohn’s disease; Effector function; Glycosylation; LC-MS/MS; Monoclonal antibodies; anti-TNFα.