In vitro protein analysis
In vitro studies were performed to investigate whether passage of adalimumab through a hollow microneedle led to protein instability, as compared to passage through a regular s.c. needle. To this end, adalimumab was subjected to the same storage conditions and ejection methods as those used in the clinical trial. Protein conformational changes were determined by second-derivative UV spectroscopy, and formation of adalimumab aggregates and particles were determined by dynamic light scattering (DLS), high-pressure size exclusion chromatography (HP-SEC), Micro-Flow Imaging (MFI), and nanoparticle tracking analysis (NTA). Results of the protein analysis are shown inTable 4 . Second-derivative UV spectroscopy showed no change in a/b ratio between conditions and time points, indicating no protein conformational changes. With DLS, no substantial differences in Z-average diameter were found. No substantial differences in the concentration of particles ≥ 2 µm were detected between conditions using MFI. NTA showed nanoparticle concentrations around the lower limit of detection (107) (data not shown), and mean sizes were found ranging from 188 to 414 nm. HP-SEC showed no differences in monomer content between conditions or between time points, and no evidence of aggregation or fragmentation. Molecular weights, based on multi angle laser light scattering (MALLS) data for the main peak, correspond to that of adalimumab reported before (20). These data show that passage of adalimumab through a hollow microneedle before storage and after storage for 4 hours at 2-8 °C does not lead to measurable protein aggregation or particle formation.