(402g) Generation of Stable Nanobubbles Following Reconstitution of Lyophilized Protein Formulations: Effects of Excipient Structure on Nanobubble Formation

Recently, reconstitution of lyophilized formulations has been shown to result in the formation of large numbers of stable bubbles approximately 150 nm in size. These nanobubbles exhibit remarkable stability, but slowly coalesce during storage over the course of days to weeks. We hypothesize that nanobubbles are formed during reconstitution as excipients dissolve around nano-sized voids present within the cake structure. Nano-sized voids may result from dehydration of regions of locally higher water concentration created during freeze concentration of solutes. These regions can be explained based on the previously reported clustering of water in polyhydroxy compounds resulting from hydrogen bonding interactions which create segregated regions of water molecules within an amorphous matrix. However, we have recently observed that lyophilization of mannitol, an excipient well known to crystallize during lyophilization, significantly increases nanobubble generation following reconstitution. Analysis of mannitol formulations using X-ray diffraction exhibited a correlation between the extent of mannitol crystallization and nanobubble generation. We believe nanobubble generation can result from dehydration of a hemihydrate crystal polymorph of mannitol to the anhydrous polymorph during drying. This dehydration process may release water back into the cake potentially generating nano-sized voids. The formation of nanobubbles following reconstitution is concerning as nanobubbles have been shown to promote aggregation of a therapeutic protein, recombinant human interleukin receptor antagonist-1. We will show that protein adsorbs to the nanobubble air-water interface resulting in significant change in nanobubble surface charge, dependent on protein net charge. Reductions in the magnitude of nanobubble surface charge following protein adsorption could be used to decrease colloidal stability of nanobubbles in protein formulation and potentially the extent of protein aggregation. Finally, we will describe the effect of metal ions leached from borosilicate glass vials on protein aggregation in nanobubble suspension. We have observed that nanobubbles stored for one week in borosilicate glass induce significantly increased monomer loss compared to suspensions stored in cyclic olefin polymer vials. Addition of a chelating agent inhibits aggregation in nanobubble suspensions further supporting the role of metal ions. The observed aggregation and particle formation in protein formulations containing nanobubbles could significantly impact both the safety and efficacy of lyophilized protein therapeutic products.