Beyond the Ice Interface: Assessment of Sugar-Modulated Intermolecular Correlations in Ice-Free Freeze-Concentrated Matrix Analogues of Monoclonal Antibodies Using SAXS

This study investigates sugar-mediated modulation of intermolecular positional correlations in monoclonal antibody (mAb) formulations upon freeze-concentration using small-angle X-ray scattering (SAXS). While previous studies have focused on solutions or dried states, experimental data on freeze-concentrated matrices (FCMs) for mAbs are still scarce, representing a key knowledge gap. Understanding the mAb FCM is key for the formulation of frozen drug substance, frozen drug product, and lyophilizates. To address this gap, we combined complementary systems: a solution concentration series of constant composition but varying total solid content, ice-free reconstituted FCM analogues, and genuine in situ frozen FCMs with varying sucrose/mAb ratios, measured within the ice-containing, non-equilibrium state. The concentration series reproduces the compositional upconcentration of all solutes that occurs upon freezing and enables tracking how protein−protein correlation features evolve with increasing crowding. A controlled cooling ramp from 20 to −40 °C probed the effect of temperature on intermolecular organization in FCM analogues and their response to cooling across Tg′. Structure factor features indicated a progressive transition from whole-molecule spacing to shorter-range, domain-level correlations with increasing concentration, converging on the distances observed in the FCM analogues and, as confirmed by cross-comparison, in the genuine in situ frozen FCM. A complementary, geometry-based coarse-grained 12-bead modelserving as an interpretive geometric frameworkreproduced the experimental correlation lengths, supporting their interpretation as sub-molecular proximity between neighboring subunits or domains. In the FCM analogues and in situ frozen FCMs, the sucrose-to-mAb ratio was a primary factor modulating intermolecular spacing, while buffer conditions had no measurable impact. Together, the FCM analogues and in situ frozen measurements provide mechanistic insight into protein packing in freeze-concentrated environments relevant for formulation stability.