5. This step must ensure that the cells are still in the exponential
growth phase, and this will increase the transfection efficiency,
baculoviral infection, and protein expression vastly.
6. Using higher amounts of DNA (up to 3 μg per 1 Â 10
6 cells)
leads to higher yields if the PEI amount is increased accordingly and the PEI:DNA ratio of 4:1 is kept stable. Further
increasing the amount of DNA will decrease cell viability due
to the required higher PEI concentration, as PEI is known to
be cytotoxic in high concentrations. This will decrease the
overall productivity.
7. Direct transfection without preforming the DNA-PEI complexes led to the highest transfection rates and protein levels
in our lab. In contrast, Shen et al. observed no differences
between direct transfection or transfection after preforming
the DNA-PEI complexes [21]. The difference might be caused
by different performance of other batches of Hi5 cells. However, a direct transfection is easier and faster. Thus, the
described direct transfection is the optimal method in our
hands.
8. Diluting with fresh media is important to keep the cells in the
exponential growth phase until the time of harvest.
9. Optimized protocols are validated and reviewed in the benchmarking study by Stolt-Bergner et al. [22].
10. For optimal expression results, the titer of the viral stock solution should be determined by a plaque assay. Additionally, the
best MOI for expression should be determined in a small-scale
expression analysis prior to scaling up by varying the MOI from
0.2 to 2.0.
11. Avidity effects can be caused by a multivalent molecule, like an
antibody, when the antigen is immobilized. For accurate determination of the kinetic constants, the orientation of the assay
must also be reverted by using anti-human IgG Fc Capture
(AHC) biosensors for the immobilization of the IgG and
Fc-tagged antibody format using KLK7 as analyte. The Fab
format cannot be analyzed in this way and needs direct chemical cross-linking to suitable tips.
Acknowledgments
We greatly thank Luciano Puzer from the Universidade Federal do
ABC, Sao Paulo, Brazil for his valuable scientific input and the
biomaterials he made available for this research project. We also
thank Daniela Gebauer, Nadine Konisch, and Anke Samuels for
their excellent technical support during the experiments. This work
was supported by the Helmholtz Protein Sample Production Facility (PSPF).
Expression of Recombinant Antibodies in Different Formats
75
growth phase, and this will increase the transfection efficiency,
baculoviral infection, and protein expression vastly.
6. Using higher amounts of DNA (up to 3 μg per 1 Â 10
6 cells)
leads to higher yields if the PEI amount is increased accordingly and the PEI:DNA ratio of 4:1 is kept stable. Further
increasing the amount of DNA will decrease cell viability due
to the required higher PEI concentration, as PEI is known to
be cytotoxic in high concentrations. This will decrease the
overall productivity.
7. Direct transfection without preforming the DNA-PEI complexes led to the highest transfection rates and protein levels
in our lab. In contrast, Shen et al. observed no differences
between direct transfection or transfection after preforming
the DNA-PEI complexes [21]. The difference might be caused
by different performance of other batches of Hi5 cells. However, a direct transfection is easier and faster. Thus, the
described direct transfection is the optimal method in our
hands.
8. Diluting with fresh media is important to keep the cells in the
exponential growth phase until the time of harvest.
9. Optimized protocols are validated and reviewed in the benchmarking study by Stolt-Bergner et al. [22].
10. For optimal expression results, the titer of the viral stock solution should be determined by a plaque assay. Additionally, the
best MOI for expression should be determined in a small-scale
expression analysis prior to scaling up by varying the MOI from
0.2 to 2.0.
11. Avidity effects can be caused by a multivalent molecule, like an
antibody, when the antigen is immobilized. For accurate determination of the kinetic constants, the orientation of the assay
must also be reverted by using anti-human IgG Fc Capture
(AHC) biosensors for the immobilization of the IgG and
Fc-tagged antibody format using KLK7 as analyte. The Fab
format cannot be analyzed in this way and needs direct chemical cross-linking to suitable tips.
Acknowledgments
We greatly thank Luciano Puzer from the Universidade Federal do
ABC, Sao Paulo, Brazil for his valuable scientific input and the
biomaterials he made available for this research project. We also
thank Daniela Gebauer, Nadine Konisch, and Anke Samuels for
their excellent technical support during the experiments. This work
was supported by the Helmholtz Protein Sample Production Facility (PSPF).
Expression of Recombinant Antibodies in Different Formats
75