Acticho sm medium

The clone stability of the CHO-K1/CN54gp140 and CHO-K1/PG9 recombinant cell lines was monitored in shake flasks (Corning) over a period of 20 passages (71–73 days) after thawing in ActiCHO SM medium (GE Healthcare Life Sciences) without selection pressure. Cells were passaged into fresh medium every 3–4 days to 2×10⁵ cells/ml. The cell concentration, viability as well as product concentration was monitored before each splitting.

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 %.