LTURE S
1
242
3. From the spinner flask, use 300-500 m1 of cell suspension at a concentration of 3-6 x lo5 cells m1-1 as inoculum forthe 1.5-1 LF 2000 bioreactorforoptimizatio~experiments (Table 5.5.1). 4. For cultures in the 12-1 NLF fermenter, apply the same parameters as optimized for the 1.5-1 KLF 2000 bioreactor (Table 5.5.1). Only the stirrer speed, which is strongly dependent on the vessel geometry, has to be re-evaluated in h 12-1 bioreactor. sfer 1500 m1of cell suspension containing 7-43 x lo5 cells ml-l from the 2000 to the NLF22 bioreactor. The dilution rateof spent to fresh medium should be l:$ and the cell density after inoculation should be about 1x lo5 cells m1-l. 6. The scale-up protocol is summarized in Table 5.5.2.
Parameters applicable to optimization in stirred bioreactors Batch Inoculation density Stirrer speed
pH PO2 Growth rate p, Oxygen supply
Scale-up of human hybridoma 4-8KH15 to the 12-1 Culture system Volume Cultivation Parameters timeParameters W ) controlled on-line measured off-line (days) 100 T-flask 1
density Cell Viability MAb conc.
Perfusion rate Circulatio~rate Cell recycle rate Oxygen supply
stirred bioreactora
_ .
11-15 500 flask Spinner
ensity
Cell KLF
14-18
NLF 18-22 000
1500
12
Viability MAb conc, Glucose pH Viability MAb conc. Glucose Lactate Cell density Viability MAb conc. Glucose Lactate
PH PO2 rpm Temperature
PR PO2 rpm Temperature
“Cells are thawed into culture medium and continuously processed from T-flasks to the NLF 22 bioreactor. At each level the parameters measured and the cumulative cultivation time required are shown. The volume of cell suspension required to proceed to the next step is given.