2D NMR assessment of structural consistency and functional relationships in monoclonal antibodies

Two-dimensional (2D) NMR at natural-abundance ¹³C has emerged as a powerful technique for generating high-resolution structural fingerprints of monoclonal antibody (mAb) higher-order structure. While increasingly applied in structural consistency and comparability assessments, the fraction of a heterogeneous mAb population required to produce a detectable change in the NMR fingerprint - and how this relates to functional impact - has not been systematically defined. In this study, we investigated the relationship between population-level structural changes detected by 2D NMR and functional consequences using antibody-dependent cellular cytotoxicity (ADCC) as a readout. A series of co-mixed mAb samples containing increasing levels of aglycosylation was prepared using homodimeric non-glycosylated heavy chain as a surrogate for aglycosylated species. Changes in 2D NMR fingerprints were evaluated using ECHOS correlation and principal component analysis and compared with FcγRIIIa binding and ADCC activity. Detectable changes in the 2D NMR fingerprint were observed only when aglycosylated mAb comprised approximately 25% or more of the population. In contrast, significant reductions in Fc receptor binding and ADCC activity occurred at lower aglycosylated fractions (approximately 10-20%). These findings demonstrate that population-level structural perturbations must exceed functional sensitivity thresholds to be detected by 2D NMR. Collectively, this work provides important context for interpreting this type of 2D NMR data in structural consistency and biosimilarity assessments, highlighting both the strengths and interpretive limits of the technique when low-level heterogeneity is present in mAb populations.