yellow. By representing the intensity of the different channels, it can be observed

that the two wavelengths are at the same position (Figure 10.4).

With the knowledge gained with this characterization, a new bioprocess called ex-

tended gene expression was designed, in order to extend the production time while

maintaining the percentage of transfected cells and therefore increase the production of

VLPs (Figure 10.5) [106]. In the extended gene expression (EGE), a medium exchange is

performed in combination with a re-transfecting of the culture every 48 hours.

As a control, in the batch standard process in transient gene expression (standard

TGE), a medium exchange before transfection was performed prior transfection,

and then VLPs were recovered at 72 or 96 hours post-transfection. Additionally, the

standard TGE was compared to another protocol called medium exchange (ME) in

which after the standard transfection a complete medium exchange was performed

every 48 hours, without any retransfection.

Since it is known that the DNA/PEI complexes are toxic to the cells, two other

protocols to reduce the amount of DNA/PEI added to the culture were studied. In the

0.5 EGE protocol after the first transfection, retransfections are performed with half of

the DNA. In the last protocol (96h EGE), more time between transfections is given;

re-transfections are performed only at 96 hours and 192 hours post-transfection.

The results of these experiments show that the viability of the batch culture (standard

TGE) starts to drop after 48 hours. For the other experiments, the viability is higher, and

the cell growth does not change significantly between the different protocols.

The percentage of GFP-positive cells drops only for the ME protocol, the con-

dition with, only medium exchange. For the other conditions in which additional

transfections were performed, the percentage of GFP-positive cells was maintained

until the end of the culture.

Regarding the production of VLPs, an increase can be observed in any of the

conditions where retransfections are performed. The best and similar results are

obtained in the EGE and in the 0.5 EGE protocols in which the production was

increased 12-fold compared with the standard batch production.

To further optimize the protocol, each of the re-transfections that in the normal

protocol are performed using the Gag-GFP plasmid was replaced with a cherry

plasmid to know the effect of a specific re-transfection on the overall production. Five

different experiments were performed evaluating the effect of each transfection.

The results show that the most important transfection is the first one. The second

and third transfections also have a significant effect on transfection efficiency and

production. When the fourth and the fifth transfections are not performed, there is not

a decrease in transfection efficiency nor production, implying that these two trans-

fections do not have a significant effect on the performance of the EGE protocol.

These results were validated in an experiment in which the EGE protocol using

half of the DNA (0.5 EGE) and the same protocol without the last two transfections

(0.5 EGE (−2)) were compared. The differences in both transfection efficiency and

production of VLPs were not statistically significant. This allowed defining an opti-

mized protocol in which the first transfection is performed using 1 ug/mL of DNA

after a medium exchange, then a medium exchange is performed every 48 hours post-

transfection, and the culture is re-transfected with half of the DNA at 48 and 96 hours

post-transfection.

Recombinant vaccines: Gag-based VLPs

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