title: Reconstitution of L-Asparaginase in Siliconized Syringes with Shaking and Headspace Air Induces Protein Aggregation.
abstract: 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.
authors.affiliation: Department of Clinical Pharmaceutics, School of Pharmaceutical Sciences, University of Shizuoka.
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Uchino
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Miyazaki
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Ohkawa
authors.forename: Takuto
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Yanagihara
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshimori
authors.forename: Mamoru
authors.initials: M
authors.lastname: Komatsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kagawa
mesh_terms: D001215:Asparaginase / Q000008:administration & dosage* / Q000737:chemistry*; D005612:Freeze Drying; D006801:Humans; D066329:Protein Aggregates; D012827:Silicone Oils / Q000737:chemistry*; D012995:Solubility; D013594:Syringes
chemical_list: D066329:Protein Aggregates; D012827:Silicone Oils; D001215:Asparaginase