Biosimilars Match Humira in Structure, Show Greater Stability Under Real-World Stress
A new analytical study underscores how advanced nuclear magnetic resonance (NMR) spectroscopy can sharpen head-to-head comparisons between Humira (adalimumab) and its biosimilars—particularly when products are challenged under real-world stress. Investigators profiled higher-order structure (HOS) for 3 lots of the Humira reference product and 2 marketed biosimilars, Yusimry (adalimumab-aqvh) and Hadlima (adalimumab-bwwd), in their commercial formulations and original container closure systems (CCSs). Under routine conditions, both 1D diffusion-filtered and 2D ^1H–^13C “methyl fingerprint” NMR spectra showed striking structural similarity across all products, a finding reinforced by chemometric analyses (ECHOS, PCA) and combined chemical-shift deviation values within ranges typical for biosimilarity assessments.
Differences emerged under photostress aligned with ICH Q1B guidance. NMR revealed new signals consistent with methionine oxidation, with the reference Humira showing the largest increase in oxidized methionine (Ox-Met) relative to the biosimilars. Expanded 2D windows pinpointed multiple oxidized sites in the reference product versus predominantly single-site oxidation in Yusimry and Hadlima. Importantly, when the methionine regions were excluded from multivariate models, partial separation between stressed and unstressed states persisted, indicating subtle but detectable light-induced conformational shifts beyond oxidation alone.
Orthogonal methods corroborated these spectral findings. Size-exclusion chromatography showed a pronounced rise in high-molecular-weight species for photo-stressed Humira (from ~0.4% to ~13.6%), whereas biosimilars exhibited only modest increases. Turbo CE-SDS further documented more low-molecular-weight species and a small purity loss after stress in the reference product, with biosimilars comparatively stable. Imaged capillary isoelectric focusing identified small shifts toward acidic variants upon photostress across all products, again most evident for Humira. Mass spectrometry provided molecular confirmation: heavy-chain mass shifts of +32 Da in the stressed reference product indicated oxidation of 2 methionine residues, compared with predominantly +16 Da in biosimilars, consistent with single-site oxidation.
Formulation and packaging appear to influence these outcomes. While all products shared a similar pH, the biosimilar formulations included histidine, an excipient often associated with oxidative protection. CCS design also mattered: autoinjector pens with smaller transparent viewing windows (eg, Yusimry) likely limited light exposure and correlated with greater photostability in this model. Notably, circular dichroism showed minimal changes in secondary structure across conditions, highlighting NMR’s sensitivity to localized, functionally relevant perturbations that bulk secondary-structure readouts may miss.
For payers and managed care decision-makers, the takeaway is twofold. First, under normal handling, biosimilars to adalimumab remain structurally comparable to the originator—a foundation for clinical interchangeability and broader access. Second, stress testing exposes formulation- and package-linked stability differences that, while not direct measures of clinical performance, may inform procurement, storage, and patient-use guidance, especially for devices more prone to light exposure. The work positions NMR as a powerful complement to established assays in confirming biosimilarity and in surfacing nuanced stability risks that can impact product quality through the distribution chain.
Reference
Baldisseri D, Luo S, Ancajas CAF, et al. NMR-based structural integrity analysis of therapeutic monoclonal antibodies: a comparative study of Humira and its biosimilars. MAbs. 2025;17(1):2551208. doi:10.1080/19420862.2025.2551208
