Cellulose acetate filter

During standard biofilm cultivations, detached biofilm biomass was quantified in the effluent in 24 h intervals. Therefore, 10 ml of the efflux liquid was collected on ice and subsequently filtered through a weighted and pre‐dried cellulose acetate filter with pore sizes of 0.22 μm (Sartorius Stedim Biotech, Göttingen, Germany) using a water jet pump. The filters were dried overnight to a constant weight at 85°C before the biomass weight was determined by differential weighting.

Agar (bacteriology grade) was purchased from AppliChem GmbH, low-melt agarose (Roti^®garose with low melting and gelling temperature) from Carl Roth GmbH & Co. KG. All hydrogels were prepared with phosphate buffered saline (PBS), pH 7.4. Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate dihydrate (Na₂HPO₄ ⋅ 2 H₂O) and potassium dihydrogenphosphate (KH₂PO₄) were purchased from Merck KGaA. The PBS buffers were prepared with ultrapure water from a Purelab Ultra water purification system (ELGA LabWater) and filtered through an 0.2 µm cellulose acetate filter (Sartorius AG) before use. 5(6)-carboxyfluorescein dihexylester was synthesized at the Institute for Biological Interfaces 1 and used as a substrate for activity assays.

Hen egg white lysozyme (HEWL) was purchased from Afilact^® (Geinberg, Germany). All other chemicals were purchased in the analysis quality from AppliChem (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Sigma-Aldrich (United Kingdom), or Honeywell Fluka™ chemicals (München, Germany) and used without further purification if not specified. All buffer solutions were filtered using a 0.45 µm cellulose acetate filter (Sartorius, Göttingen, Germany) and those for chromatographic applications were further degassed in an ultrasonic chamber (5510E-DTH, Bransonic, Missouri, United States).
Humira was produced in a recombinant monoclonal Chinese hamster ovary K1 (CHO_K1) cell line using Geneticin G-418 Sulphate (Gibco, United Kingdom) as the selection system. Fed-batch fermentations with chemically defined ActiPro™ medium (HyClone™, Austria) supplemented to 8 mM L-Glutamine were realized in DASGIP^® Parallel Bioreactor System (Eppendorf, Germany). Fermentation parameters were set to 60 rpm stirrer speed, 37°C, 60% dissolved oxygen, and 3 s L/h gas flow rate, and pH 7.2 was controlled by CO₂ and 7.5% sodium bicarbonate addition. A glucose concentration of at least 2 g/L was maintained by a 10% glucose feed. Harvesting of the fermentation broth was performed on day 7 with maximum cell densities of 11 × 10⁶ cells/mL.

Freeze-dried NPs (5 mg) were dissolved in 1 mL of dichloromethane. Then, 3 mL of PBS pH 7.4 were added to extract the FITC-albumin and the organic solvent was evaporated at RT under stirring (1,500 rpm for at least 1 h; RW20DZM, Janke&Kunkel, IKA-Labortechnik, Staufen, Germany). The aqueous solution was filtered (cellulose acetate filter, porosity 0.2 μm, Sartorius) to remove the polymer residues and spectrophotometrically analyzed at 492 nm to evaluate the FITC-albumin concentration. Drug loading was expressed as mg of FITC-albumin encapsulated/100 mg of NPs and as encapsulation efficiency (EE%), i.e. the percentage of encapsulated drug related to the initial amount of drug used in the preparation.

At the end of the experiment, particulate organic carbon (POC) and particulate organic nitrogen (PON) were measured after filtering onto pre-combusted (15 h, 500°C) GF/F filters (pore size ~ 0.6 μm, Whatman). The amount of seawater filtered ranged between 200–300 mL and was dependent on the biomass in the treatments. Filters were stored at -20°C and dried for > 12 h at 60°C. Analysis was performed using a Euro Elemental Analyzer 3000 CHNS-O (HEKAtech GmbH, Wegberg, Germany). At the end of the experiment, samples to determine biogenic silica (BSi) were filtered through a cellulose acetate filter (Sartorius, 0.6 μm) and stored at -20°C. The dried filters were submerged in 0.2 M NaOH at 95°C for 45 minutes, cooled in an ice bath for 15 minutes, neutralized with 1 M HCl according to [66 (link)] and analyzed colorimetrically for silicate using standard spectrophotometric techniques [67 ]. Contents of POC, PON and BSi were corrected for blank measurements and normalized to filtered volume and cell densities to obtain cellular quotas. Production rates of POC, PON and BSi were calculated by multiplying the cellular quotas with the respective growth rate.