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.