Purpose: Certain monoclonal antibodies (mAb) are observed to be prone to aggregation and exhibit increased solution viscosity under typically high formulation concentrations, (generally ≥ 100mg/mL). Unlike the traditional biophysical methods which probe average conformational/colloidal properties of proteins, we aim to investigate conformational stability of these multi-domain proteins at a residue-level. Solution nuclear magnetic resonance spectroscopy (NMR) provides insights into conformational states of these proteins at a residue level, otherwise invisible in a static crystal structure.
Methods: In our study, fragments of mAbs (Fab and Fc domains) were generated by enzymatic cleavage using IdeS, a highly specific IgG cleaving enzyme. High resolution backbone amide-based [15N-1H] and methyl group-based [13C-1H] 2D NMR spectra were obtained for molecular fingerprinting of Fc domain. The thermal stability of some of the intact mAbs and their cleaved components were assessed by Differential Scanning Calorimetry.
Results: A set of selected Pfizer mAbs of isotype IgG2 of varying aggregation propensities were used in our studies. Furthermore, the methodology was also extended to an intact bispecific protein to probe the aggregation behavior of the protein under different buffer conditions. Based on 2D NMR based molecular fingerprinting, Fc domains of aggregation prone mAbs seem to have unique molecular fingerprints (conformation) as shown in Figure 1.
Conclusion: The molecular fingerprinting of mAbs and mAb-related proteins as obtained by 2D NMR methods suggests the presence of certain conformational states that can act as early transients for overall aggregation of mAbs.