Fed-batch optimization of transient scFv-Fc-RNase protein production in HEK293-6E cells. The vector pCMV-oriP1-scFv-Fc-RNase-4E3 was transiently transfected into HEK293-6E cells in 25 mL scale in shake flasks. One volume of fresh medium and 0.5% (w/v) tryptone TN1 was fed 48 h after transfection to all groups (group A, B, C). After 3 days two groups (group B, C) were fed with glucose and one group with additional 3.75 mM valproic acid (VPA, only group C). A) Volumetric yields of scFv-Fc-RNase protein were measured from supernatant samples taken from day 2 to 8 after transfection. B) ScFv-Fc-RNase was purified by protein A affinity chromatography from production supernatants harvested 8 days after transfection. The yield was calculated from total amount of purified protein and the final production volume. Protein recovery was determined as ratio of scFv-Fc-RNase after and before purification. C) Cell number, D) viability and and transfection efficiency (GFP co-transfection), E) Glucose and lactate as well as F) L-glutamine and glutamate were measured during the production until day 7. All groups were performed in independent triplicate samples. Mean values as well as standard deviations are given.