Reconstitution of L-Asparaginase in Siliconized Syringes with Shaking and Headspace Air Induces Protein Aggregation.

The aim of this study was to characterize protein aggregation during reconstitution of a highly concentrated solution of lyophilized L-asparaginase (L-ASP). The effect of the preparation method on L-ASP aggregation using siliconized or non-siliconized syringes and the effect of storage after preparation were evaluated by far-UV circular dichroism spectroscopy, Raman microscopy, flow cytometry, and flow particle image analysis. To investigate the effect of syringe type in combination with shaking and headspace air on L-ASP aggregation, four kinds of L-ASP in 5% glucose solutions were prepared (in the presence or absence of silicon oil and headspace air). Slight differences in L-ASP secondary structure were observed between the siliconized and non-siliconized syringe systems before shaking. Large numbers of sub-visible (0.1-100 µm) and submicron (0.1-1 µm) particles were formed by preparation with siliconized syringes and the combination of shaking and headspace air. The number of aggregated particles was not decreased with increased storage time. The Raman microscopy, flow cytometry and flow particle image results suggested that L-ASP interacted with silicone oil, which induced aggregation. Nevertheless, sub-visible and submicron particles were also formed with non-siliconized syringes. However, using non-siliconized syringes, the number of aggregated particles decreased with storage. No changes in particle character were observed before or after shaking with headspace air in non-siliconized syringes, indicating that soluble aggregates formed and dissolved with storage. Silicone oil in syringes, in combination with shaking and headspace air, strongly affected the aggregation of lyophilized L-ASP formulations during preparation.

Particle condition change in emulsion admixture evaluated by in situ flow particle imaging analysis.

We evaluated the particle state change in emulsion admixtures using in situ flow particle imaging analysis (FPIA). Ropion® intravenous (flurbiprofen axetil: Ropion®) served as the model emulsion formulation. A binary mixture of Ropion® and normal saline (NS), and a ternary admixture of Ropion®, NS, and Gaster® injection (famotidine: Gaster®) or Primperan® injection (metoclopramide hydrochloride: Primperan®) were prepared and the change in emulsion particle state was analyzed using FPIA under in situ condition. The effect of storage on pH change and the chemical stability of flurbiprofen axetil were also investigated. In Ropion®, various particle images (mean diameter: 2.4 µm) were obtained. From our analysis of changes in scattergrams and particle images, changing behaviors of emulsion particles as a function of storage time depended on the systems of admixture samples. In Ropion®/NS and Ropion®/Gaster®/NS systems, mean particle size and particle number increased with lengthening storage time; however, these values were dramatically increased beyond 6 h in the Ropion®/Primperan®/NS system, corresponding to a decrease in measured pH. The decomposition of flurbiprofen axetil due to incompatibility was not observed in all systems. Detailed information on the change in emulsion particle state was obtained using FPIA, indicating that this method is useful to evaluate state changes in emulsion admixtures under in situ condition.