Methods to study device induced aggregation of proteins

Abstract: This work developed a method for the investigation of how medical devices, such as syringes and needles, affect the formation of subvisible particles in a protein drug without excipients. Protein-based pharmaceuticals are well known to be sensitive to different kinds of stress, and exposure to stress during the use of this device is unavoidable. To study this phenomenon, an in-house built probe for the operation of syringes with a texture analyser was created. Mechanical stress was modulated by varying the expulsion speed, and related forces were captured by the texture analyser. The formation of particles was evaluated by dynamic light scattering(DLS), size exclusion chromatography(SEC) and flow imaging microscopy by FlowCAM. The insulin was found to be primarily present in its hexamer form, with a hydrodynamic diameter of 5.6 nm. FlowCAM showed increased concentration of particles in both siliconized and non-siliconized syringes after mechanical stress. The increase was associated with protein aggregates in the non-siliconized syringe, while increase in the the siliconized was mainly associated to silicone oil droplet formation. The particles were categorized by sphericity using circle fit and aspect ratio, with spherical cutoff at 0.85. The siliconized syringe had no increase in protein aggregates after stress and displayed a substantial loss of native protein (2.5 %) according to SEC. This concedes with findings from other studies and might be due to oil interface adsorption. Although, the size distribution from FlowCAM yielded particle counts, which were theoretically too low to account for this loss alone.