Anti clumping agent

The recombinant CHO-EpoFc cell line was established as previously described (Lattenmayer et al., 2007 (link)) and was later adapted to growth in serum-free and l-glutamine free CD CHO medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 0.096 μM MTX and 1 ml anti clumping agent (Gibco, Invitrogen, Carlsbad, CA, USA) per 500 ml medium (Taschwer et al., 2012 (link)). Cell cultures were cultivated in conical flasks with a working volume of 30 ml at 37 °C in a humidified atmosphere containing 7% carbon dioxide and constant shaking at 140 rpm.

Cell culture was performed with CHO^BRI cells (Biotechnology Research Institute, QC, CA) adapted to grow in a commercial serum-free, CD-CHO growth media (Life Technologies Inc., ON, CA) supplemented with Gibco^® GlutaMAX^™ (10 mL per 1 L CD-CHO), HT supplement (10 mL per 1 L CD-CHO), Pluronic F-68 non-ionic surfactant (10 mL per 1 L CD-CHO), and anti-clumping agent (10 mL per 1 L CD-CHO) (Life Technologies, Inc., ON, Canada). Both GlutaMAX^™ and HT supplement were added to the media prior to UV irradiation, while Pluronic F-68 and anti-clump were added prior to inoculation (after UV irradiation). The parental culture was incubated at 37°C and 5% CO₂ with agitation at 120 rpm. Erlenmeyer flasks (125 mL non-pyrogenic polycarbonate, Corning Inc., NY, US) with a vented cap were used for the parental and experimental cultures, and the working volume of the flasks was 30 mL. When the mother flask reached a viable cell density of 2 to 3 × 10⁶ cells/mL, the experimental cultures were inoculated with a seeding density of 0.2 × 10⁶ viable cells/mL. Experimental cultures for each treatment group were inoculated and cultured in triplicate. Cell density and viability were monitored daily for up to eight days using a haemocytometer by trypan blue exclusion. When the cells were no longer viable, the cell pellet and supernatant were separated by centrifugation at 500 g for 10 min.

CHO-S suspension cells (Life Technologies) were grown in CD CHO medium supplemented with 8 mM L-glutamine and 2 μL/mL anti-clumping agent (Life Technologies). Cells were expanded in Corning vent cap shake flasks (Sigma-Aldrich, St. Louis, MO) in a humidified incubator at 120 rpm (25 mm orbit), 37 °C, and 5% CO₂.

Suspension and serum-free adapted CHO-DUKXB-11 cells were grown in DMEM:Ham’ F12 (1:1) supplemented with 4 mM l-glutamine and protein-free additives without growth-factors (CHO-DUKXB-11). All other cell lines were cultivated in CD CHO media (Life Technologies) supplemented with 8 mM l-glutamine (CHO-K1-8 mM and CHO-S) or without (CHO-K1-0 mM) and 1:500 anti-clumping agent (Life Technologies). Recombinant CHO-DUKXB-11 cells expressing an erythropoietin-Fc fusion protein were grown in suspension in CD CHO media with 0.019 μM methotrexate and without l-glutamine supplementation (Taschwer et al., 2011 (link)). No defined growth factors such as Insulin or IGF were used as additives in this study.
All cell lines were cultivated in suspension in Erlenmeyer shake flasks in 50 ml volume at 140 rpm in a shaking incubator (Kuhner, Switzerland) in a humidified atmosphere (90%) conditioned with 7% CO₂.