Fed-Batch Induction Experiments
The fed-batch induction experiments were performed in 24-well deep well plates (Axygen Scientific) using Duetz covers. The cultures were seeded at 2 x 105 cells/mL in 3.5 mL CD CHO medium supplemented with 100 µg/mL hygromycin B, 4.5 µg/mL bleocin, and 4 mM glutamine, and incubated at 37°C, 5% CO2 and 225 rpm in a humidified incubator (Kuhner shaker). The experiment was initiated by adding the AIP stock solution (1.65 mM) to obtain medium concentrations of 0, 1, 2, 3 and 4 µM. Starting on day 1, the fed-batch regime was initiated with a 280 µL sample taken daily from the culture. This volume was then replaced with 140 µL CD CHO Efficient Feed A (Gibco), 70 µL amino acid mixture A [10 mM L-cystine disodium salt hydrate (MP Biomedicals, Illkirch, France), 15 mM L-tyrosine disodium salt (Sigma, St. Louis, MO) and 10 mM aspartic acid (Gibco) dissolved in 0.1 N HCl] and 70 µL amino acid mixture B [10 mM L-glutamic acid (Sigma) and 75 mM L-asparagine (Sigma) in 0.1 N NaOH]. Every two days, 150 µL of the removed culture volume was mixed with 150 µL of DPBS and used for Cedex analysis at a 1:2 dilution. As above, when cultures reached a concentration of greater than 2 x 106 cells/mL, a 100 µL aliquot was taken to determine cell count and viability at a 1:3 dilution with DPBS. Similar to the batch cultures, the remainder of the culture sample was centrifuged and the supernatants were retained for IgG quantitation by ELISA. Each AIP medium concentration was evaluated in duplicate cultures during a run and the entire fed-batch induction experiment was repeated (for a total of 3 times).