Immunogenicity of protein aggregates of a monoclonal antibody generated by forced shaking stress with siliconized and nonsiliconized syringes in BALB/c mice.

OBJECTIVE: In this study, we aimed to investigate the immunogenicity of protein aggregates of monoclonal antibodies (mAbs), generated by forced shaking stress with siliconized and nonsiliconized syringes in a mouse model. METHODS: Samples were filled in siliconized and nonsiliconized syringes with shaking and headspace air. Characterization studies were performed using high-performance size-exclusion chromatography, nanoparticle tracking analysis, flow cytometry, micro-flow imaging and resonant mass measurement. The samples (10 or 100 µg) were subcutaneously injected into BALB/c mice for 21 days, and the anti-drug antibody (ADA) concentrations were monitored. KEY FINDINGS: In samples shaken with siliconized syringes [SO (+)], large amounts of submicron and subvisible protein aggregates were formed by interactions with silicone oil droplets. The characteristics of protein aggregates differed between the mAb solution and shaken samples, which strongly indicates that silicone oil accelerates protein aggregation. When administered at low doses, the ADA concentration in all samples increased with repeated injections, and SO (+) induced the highest immunogenicity. However, when administered at high doses, ADA concentration decreased following prolonged repeated administration for tolerance. CONCLUSIONS: These results indicated that mAb protein aggregation induced immunogenicity in mice, and SO (+) induced higher immunogenicity than samples shaken with nonsiliconized syringe.

Modulation mechanism of the stratum corneum structure during permeation of surfactant-based rigid and elastic vesicles.

We evaluated the interaction between human stratum corneum (SC) and surfactant-based rigid or elastic vesicles during permeation using synchrotron X-ray diffraction to obtain the mechanism action of surfactant-based vesicles for enhanced skin permeation. The effects of vesicle elasticity on the interaction with SC were also investigated. Changes in the small-angle X-ray diffraction peaks of the human SC after buffer control and vesicle application were monitored. In the small-angle region, the control as the citrate buffer induced no significant changes of diffraction peaks for the lamellar structure of short periodicity phase (SPP), which is observed as a main peak in the human SC. Application of rigid vesicles resulted in small changes in the diffraction peaks attributed to lamellar phase of SPP. After application of elastic vesicles, however, a large shift to smaller angles due to swelling of the lamellar phase of SPP was clearly observed from the intensity difference profiles. All peaks due to the vesicles were still observed after 2h of application for all formulations, indicating that both vesicles interacted with the SC while maintaining their structures. These results strongly suggest that vesicles affect the lamellar phase of SPP of the intercellular lipids in the SC during permeation.

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.