Freestyle tm max reagent

CHO-S suspension cells (Life Technologies, Thermo Scientific, Rockford, IL ) were grown in CD CHO medium (#10743029, Life Technologies) supplemented with 8 mM L-glutamine (#LONZ17-605F, Lonza Group AG, Basel, Switzerland) 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. Transfection was performed essentially as previously described [13] . In brief, 3x10 7 (recombinant human erythropoietin; rEPO) or 5x10 7 (empty vector and α1AT) cells were transfected using FreeStyle TM MAX Reagent (Life Technologies) in 30 mL (EPO) or 50 mL (empty vector and α1AT) complete CD CHO medium without anti-clumping agent according to manufacturer's instructions. Transfected cells were incubated in Corning vent cap shake flasks (Sigma-Aldrich) at 120 rpm (25 mm orbit), 37°C, and 5% CO2. 3 hours post-transfection, anti-clumping agent was added to reach a 2 µL/mL final concentration. Viable cell density (VCD) and viability were measured every day (day 0 -3) and supernatant samples were obtained from day 1 to day 3.

Cells were seeded in 250 mL Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies) and transfected with 75 μg of A1AT-Glul-ST6GAL1 plasmid or 75 μg of C1INH-Glul-ST6GAL1 plasmid (Suppl. Fig. 1) using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer's recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies. Two days after transfection, cells were transferred into 60 mL CD CHO medium lacking L-glutamine (Life Technologies) and supplemented with 1μL/mL anti-clumping agent and 0 μM, 10 μM, 30 μM or 50 μM MSX (EMD Millipore, Billerica, MA).
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). The cells were passaged in fresh selection medium every 2-3 days until viability and doubling time reached stable values. Polyclonal cell lines (pools) were seeded in duplicates at ~1 x 10 6 cells/mL with corresponding MSX concentrations. Cell densities and viabilities were determined once per day and supernatants of the pools were harvested three days after seeding and pooled within duplicates for purification of rhA1AT and rhC1INH.

For transient expression of rituximab/EPO, cells were seeded in Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2 and transfected with 75 μg of rituximab or EPO encoding plasmid for each flask using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). To purify rituximab and EPO, the supernatants of the transfected clones were harvested three days after transfection and pooled within duplicates.