Acticho p

The suspension CHO cell line producing a 150 KDa fully humanized tumor necrosis factor receptor Fc-fusion protein from Research Working Cell Bank (RWCB) of Aryogen Pharmed (Alborz, Iran) was used. The original source of CHO DG44 ordered from Thermo Fisher Scientific, /Gibco, USA (Catalog no: A10971-01). The culture medium was ActiCHO P (GE Healthcare) supplemented with 6 mM L-glutamine (Lonza, Verviers, Belgium) and related feeds (feed A and B, GE Healthcare) at the moment of preparation as recommended by the supplier. Cells were propagated using 500-ml baffled shake flask containing 100-ml culture media incubated on an orbital shaker with 75 RPM (30 mm amplitude of rotation, Witeg, Germanny) at 37°C with 5% CO2.

A CHO DG44 cell line (licensed from Cellca GmbH, Laupheim, Germany) was used as a model cell line. The cells were thawed and propagated in ActiCHO™ SM medium (GE Healthcare) supplemented with 6 mM L-glutamine and 30 nM methotrexate (both Sigma-Aldrich). In routine culture and batch experiments, cells were inoculated at a cell concentration of 2 × 10⁵ cells/mL, using a working volume of 35 mL in 125-mL Erlenmeyer shake flasks (Corning). Cultures were grown in an ISF1-X incubator shaker (Kuhner) at 37 °C, 140 rpm, 7 % CO₂, and 90 % humidity. The cells were passaged every 3–4 days. Adaptation to new media was done through at least five consecutive passages, until a stable growth rate had been achieved. The media investigated were CD CHO, CD OptiCHO™, CD FortiCHO™ (all Life Technologies), Ex-Cell™ CD CHO (Sigma Aldrich), ProCHO™5 (Lonza), BalanCD™ CHO Growth A (Irvine Scientific), Cellvento™ CHO-100 (EMD Millipore), and ActiCHO™ P (GE Healthcare). After adaptation to the new media, batch cultures were started and sampled daily as described below. Cultures were terminated once the viability dropped below 60 %.

Suspension cultures of two CHO-K1-derived cell lines (referred to as cell lines A and B) expressing the same IgG1 monoclonal antibody were maintained in shake flasks before inoculating the bioreactors. Stocks were revived in commercially available basal medium (ActiCHO P, GE Healthcare, UK), supplemented with 8 mM L-Gln and 5 mg/l insulin. The cells were sub-cultured every 3–4 days with a seeding density of 0.3 · 10⁶ cells/ml and were grown in shake flasks of different scales. The shake flasks were incubated at 37 °C with humidified air containing 5 % CO₂ and agitated at 100-rpm orbital shaking.