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.