5. Pipette the DNA mix directly to the prepared cells and mix
gently (see Note 7).
6. Immediately pipette 4 mL PEI of the 1 mg/mL PEI stock
solution to the cells and mix gently.
7. Incubate the culture at 27
C and 110 rpm for 3 up to 5 h.
8. Add 400 mL fresh EX-CELL 405 (see Note 8).
9. [Day 1–3, Tuesday to Thursday] Take samples daily, monitor
cell numbers and viability, and determine transfection efficiency
in the cytometer. If cells reach a density above 3 Â 10
6 c/mL,
adjust the concentration back to 2 Â 10
6 c/mL by adding fresh
culture medium.
10. [Day 3, Thursday] Harvest secreted target proteins by centrifuging the cell suspension 15 min at 1000 Â g and store the
supernatant at 4
C after sterile filtration (0.45 μm) until
purification.
Fig. 3 Transient antibody production in HEK293-6E cells. (a) Schematic vector map of the mammalian
HEK293-6E expression vector with exemplary expression cassette (found in pTT5 or pFlpBtMII [2]). (b)
Schematic illustration of corresponding gene expression cassettes used for the production of recombinant
antibodies in the scFv-Fc, Fab, and IgG format. (c/d) Monitoring of growth, viability, and transfection efficiency
(GFP-positive cells) over time. Volume doubling and supplementations at 48, 72, and 96 h post-transfection
are indicated by black arrows. Mean values are presented
Expression of Recombinant Antibodies in Different Formats
67
gently (see Note 7).
6. Immediately pipette 4 mL PEI of the 1 mg/mL PEI stock
solution to the cells and mix gently.
7. Incubate the culture at 27
C and 110 rpm for 3 up to 5 h.
8. Add 400 mL fresh EX-CELL 405 (see Note 8).
9. [Day 1–3, Tuesday to Thursday] Take samples daily, monitor
cell numbers and viability, and determine transfection efficiency
in the cytometer. If cells reach a density above 3 Â 10
6 c/mL,
adjust the concentration back to 2 Â 10
6 c/mL by adding fresh
culture medium.
10. [Day 3, Thursday] Harvest secreted target proteins by centrifuging the cell suspension 15 min at 1000 Â g and store the
supernatant at 4
C after sterile filtration (0.45 μm) until
purification.
Fig. 3 Transient antibody production in HEK293-6E cells. (a) Schematic vector map of the mammalian
HEK293-6E expression vector with exemplary expression cassette (found in pTT5 or pFlpBtMII [2]). (b)
Schematic illustration of corresponding gene expression cassettes used for the production of recombinant
antibodies in the scFv-Fc, Fab, and IgG format. (c/d) Monitoring of growth, viability, and transfection efficiency
(GFP-positive cells) over time. Volume doubling and supplementations at 48, 72, and 96 h post-transfection
are indicated by black arrows. Mean values are presented
Expression of Recombinant Antibodies in Different Formats
67