In vitro protein analysis
Adalimumab 100 mg/mL pre-filled pens or syringes (depending on
availability) of the same batch and expiration date were pooled. Storage
containers were: (1) syringe only, (2) Verex 2 ml clear glass vial
(Phenomenex, Torrance, CA, USA); (3) syringe with a MicronJet600. For
condition (1) a capped regular needle was attached during storage to
prevent evaporation. Samples were measured immediately (to assess the
effect of repackaging), or after storage for four hours at 4°C (to
assess in-use stability). Directly before analysis the samples were
ejected from the syringe into a glass vial and subsequently diluted to
10 mg/mL or 1 mg/mL with solvent. The solvent consisted of Milli-Q water
with 1.2 g per 100 mL mannitol (Sigma, St Louis, MO, USA) and 100 mg/100
mL polysorbate 80 (Sigma, St Louis, MO, USA), and was filtered through
an Anotop 10 mm, 0.1 µm syringe filter (Whatman, Maidstone, UK) before
use. For NTA optimization, the solvent was made without polysorbate 80.
Experiments were performed at room temperature, and in a dust free
cabinet whenever possible. Changes in protein conformation were
determined by second-derivative UV spectroscopy. The formation of
adalimumab aggregates and particles was determined by dynamic light
scattering (DLS), high-pressure size-exclusion chromatography (HP-SEC),
Micro-Flow Imaging (MFI), and nanoparticle tracking analysis (NTA) as
described (20) and summarized below.