The fed-batch strategy was designed to expand a culture within a single HybridBag from 1 to 7 L, thereby reducing the need for a large initial inoculate to be produced in many shaker flasks.
The purpose of this study was to demonstrate the flexibility and adaptability of this system for large-scale cell culture using a complex fed-batch protocol. A CHO-S cell inoculation culture was grown up to a final cell density of 1x106 cells/mL in shaker flasks in CHO-S media (Invitrogen) at 37ºC, 8% CO2, with a shaker speed of 80 rpm.
The HybridBag culture was established using 1 L of CHO-S media preheated to 37ºC and was inoculated with CHO-S cells to a final concentration of 5x105 cells/mL. The culture was gently stirred using the HybridBag propellers at 30 rpm (30% max speed), which prevented cells from settling and achieved culture aeration. An overlay in the headspace was created with a mixture of 46% air, 46% N2, and 8% CO2, with a flow of 100 mL/min.
The integrated WIDGET™ dO2 probe was used to measure dO2 concentration, which was maintained by the CellMaker PLUS Controller at 30% air saturation by adjusting air/N2 ratios.
Once the cell density had reached 1x106 cells/mL, the culture was diluted by the addition of 1 L of fresh, preheated CHO-S media to give a new culture volume of 2 L and growth was monitored.
D-glucose was determined using a hand-held blood-glucose analyzer, and the culture concentrations were maintained between 1,000 and 1,500 mg/L by the addition of fresh D-glucose from a 100 mM stock solution. In addition, fresh L-glutamine was added to 0.2 mM L-glutamine final concentration every 72 hrs.
Once the 2 L cell culture in the HybridBag had reached a cell density of >8x105 cells/mL, culture aeration was switched from the headspace to the sparge tube; this increased the efficiency of aeration, which was necessary for optimal growth of the expanding culture.
The combination of the integral propellers and airlift also increased the mixing of the culture and eliminated cell settling, even in the corners of the HybridBag where settling might have been expected.
Antifoam (Sigma SE-15, 10% v/v) was routinely added to all cultures and had no impact on cell density or viability at the concentrations used. Antifoam preparation (1 part antifoam plus 2 parts PBS) was added to the HybridBag and shaker flask control to achieve a concentration of 1–5 ppm every 72 hrs.
The 2 L of expanded culture in the HybridBag was then diluted with a further 2 L volume of fresh CHO-S media once the cell density saturated at >1.5x106 cells/mL, to give a new culture volume of 4 L. The culture was maintained in the HybridBag, using this volume expansion/feeding strategy, to a final working volume of 7 L, at which point the culture was grown to saturation, achieving a final cell concentration of 3.8x106 cells/mL.
Throughout the growth of the culture, cell counts and viability were determined using a hemocytometer and trypan-blue staining. Viabilities were determined to be >95% in the HybridBag culture.