The ambr™ system, with vessel dimensions of 28 mm × 16 mm × 58 mm, maintained the culture temperature at 37 ☌, agitation at 1,400 rpm, and DO at 30 % of air saturation. The culture material taken from the newly inoculated 2-L bioreactors for each cell line was used to seed six ambr™ vessels at 14 mL working volume. When glucose dropped below 4 g/L, the culture received a concentrated glucose feed consisting of a 500 g/L glucose stock solution to increase culture glucose by 12 g/L. On day 3, a single nutrient feed consisting of a proprietary blend of chemically-defined ingredients was added to all cultures, increasing the culture volumes by 20 %. On day 3, the culture temperature was shifted to 35 ☌ for cell lines 1 and 2, and to 33 ☌ for cell lines 3 and 4. Culture temperature was maintained at 37 ☌ initially for all cell lines from days 0–3. Separate mass flow controllers were used for air and oxygen, and DO was controlled in cascade mode by sparging with air and then with the required amount of supplemental oxygen. At the time of inoculation, each bioreactor culture started with a constant 10 mL/min air sparge, corresponding to 0.0067 vvm of air flow. Culture pH was maintained by addition of 1 M Na 2CO 3 to increase pH, or by sparging of CO 2 gas to decrease pH. The cultures were maintained with setpoints for pH at 7.0, dissolved oxygen at 30 % of air saturation, and agitation (via single pitched-blade impeller with 45 mm diameter and impeller power number 1.5) at 350 rpm. The bioreactor process setpoints were controlled by DCUs (B. Each identically-configured stirred-tank bioreactors had a maximum working volume of 2 L, a vessel diameter of 0.13 m, and vessel height of 0.25 m. The other culture was inoculated at 2.0 L, from which 0.5 L was removed to seed the parallel ambr™ and shake flasks cultures after inoculating the 2-L bioreactor. The transfected cells were passaged every 3–4 days in proprietary chemically-defined media (derived from Ham’s F12/Dulbecco’s Modified Eagle’s Medium) containing selective pressure (methotrexate for cell lines 1 and 2, and methionine sulfoximine for cell lines 3, 4, 5, and 6).įor each cell line tested, two glass stirred-tank bioreactors (Applikon, Foster City, CA) were seeded at ~1.7 × 10 6 cells/mL in a proprietary, chemically-defined medium. To generate these cell lines, the host cells (CHO DHFR- or CHO-K1) were transfected with a DNA plasmid encoding genes for the appropriate selection marker, light chain, and heavy chain. Cell lines 5 and 6 are different clones that secrete recombinant monoclonal antibody C, and were also derived from a CHO-K1 host that utilizes the glutamine synthetase selection marker. Cell lines 3 and 4 are different clones that secrete recombinant monoclonal antibody B, and were derived from a CHO-K1 host that utilizes the glutamine synthetase selection marker. We also cultured two additional CHO cell lines in parallel ambr™ and 2-L bioreactors to compare different DO control strategies.Ĭell lines 1 and 2 are different clones that secrete recombinant monoclonal antibody A, and were derived from a CHO dihydrofolate reductase-deficient (DHFR-) host as described previously (Guo et al. We simultaneously cultured four representative CHO cell lines in the ambr™ system, 2-L bioreactors, and shake flasks, and we assessed (1) online controls (temperature, DO, and pH) provided by the ambr™ and 2-L bioreactor systems (2) off-line pO 2, pH and pCO 2 profiles and (3) culture performance (growth, metabolism, productivity, and product quality) in the three culture systems. Here we conducted a more extensive evaluation of the ambr™ system to determine its ability to support cell culture process development. When they cultured three CHO cell lines in fed-batch mode in the ambr™ system and compared the performance to historic data from 7-L bioreactors, they observed comparable viability and antibody production profiles (Lewis et al. ( 2010) observed reproducible growth profiles for a Chinese hamster ovary (CHO) cell line cultured simultaneously in all 24 vessels under identical process conditions.
#Nova bioprofile 400 gln sensitivity trial#
In a recent trial run for the ambr™ system, Lewis et al. The newly developed advanced microscale bioreactor (ambr™) system uses 24 or 48 single-use, pre-sterilized, stirred-tank bioreactors on an automated workstation that enables pH and dissolved oxygen (DO) control for each individual vessel (Bareither and Pollard 2011). Although shake flasks provide higher throughput than bench-top bioreactors, they do not offer the environmental control capabilities of a bench-top bioreactor. Scale-down systems such as shake flasks and bench-top bioreactors are typically used to develop cell culture processes for clinical and commercial manufacturing. Several scale-down cell culture systems have been developed in recent years to reduce the timelines and costs associated with cell culture process development (Bareither and Pollard 2011 Betts and Baganz 2006 Kondragunta et al.