Heterogeneous nucleation-controlled particulate formation of recombinant human platelet-activating factor acetylhydrolase in pharmaceutical formulation.

Clinical lots of recombinant human platelet-activating factor acetylhydrolase (rhPAF-AH) were prepared in a lyophilized formulation. After reconstitution with sterile water for injection to form an aqueous solution (10 mM sodium citrate, 7.5 w/v% sucrose, and 0.1 w/v% Pluronic-F68, pH 6.5), a few visible, slowly growing particles formed consistently within hours at room temperature. To investigate the mechanism of this phenomenon, immediately after reconstitution, all protein aggregates and exogenous particles were removed by filtration. During 20 days incubation at room temperature, no visible aggregates formed in these filtered samples. In contrast, when nano-sized hydrophilic silica particles were added, they seeded rapid and extensive aggregation of rhPAF-AH. This effect was exacerbated in solutions containing a lower Pluronic-F68 concentration at 0.01%. Aggregation occurred even under conditions where rhPAF-AH adsorption was reversible, and induced no detectable changes to protein secondary and tertiary structures. Decreasing the extent (e.g., adding Pluronic-F68) or affinity (e.g., increasing solution pH) of rhPAF-AH adsorption on nano-sized silica particles was found to be effective at reducing aggregation. Accelerated aggregation was not observed when rhPAF-AH formulation was seeded with aggregated rhPAF-AH. These results show that rhPAF-AH aggregation proceeds through a heterogeneous nucleation-controlled mechanism, where exogenous particles present in solution serve as seeds on which rhPAF-AH adsorb, nucleate, and grow into large aggregates. Copyright 2004 Wiley-Liss, Inc.