Contradictory Effects of Different Freezing Rates on Visible Bubbles and Reconstitution Time during the Reconstitution of Freeze-Dried High-Concentration Monoclonal Antibody Formulations

The freezing rate is a critical factor governing ice nucleation and growth during freeze-drying, which directly determines the microvoid architecture of the freeze-dried cake and its reconstitution properties. This study systematically evaluates the effects of controlled freezing methods on reconstitution time, formation of visible bubbles (VBs), and long-term stability of high-concentration monoclonal antibody (mAb) formulations─addressing a pivotal challenge in freeze-drying process development. Solid-state microstructure was analyzed using scanning electron microscopy and mercury intrusion porosimetry. Paradoxically, accelerated freezing rates had divergent effects on reconstitution: they shortened the reconstitution time but increased the level of generation of VBs. Nonetheless, protein stability─as assessed by size exclusion chromatography and ion-exchange chromatography, remained unaffected across different freezing rates. Importantly, employing an intermediate freezing rate significantly reduced VB formation and shortened the reconstitution time for high-concentration mAb formulations. These results underscore freezing rate modulation as an essential strategy for optimizing the reconstitution performance of freeze-dried high-concentration biologics without compromising stability, offering valuable insights for industrial pharmaceutical development.