well as supplements of plant origin or synthetic nature and facilitates switching to

efficient animal-derived component-free production processes.

Importantly, not all culture media can be used for PEI-mediated transient

transfection [46,75–77] because of the presence of anti-clumping agents such as

heparin or dextran sulfate polyanions (frequently added to suspension culture media

formulations to prevent cells from aggregating) in some formulations.

10.4.2

CELL LINES/PLASMIDS

A key point in the recombinant protein expression is the stability of the plasmid

within the cell. To increase plasmid persistence, a successful strategy developed has

been the constitutive expression of the large T antigen of simian virus 40 (SV40) in

293-T, CHO-T or CAP-T cell lines, increasing therefore the replication of vectors

containing the SV40 origin of replication [78–82].

Another genetic element engineered for the optimization in mammalian cells, is

the addition of EBNA-1 gene of the Epstein –Barr virus in conjunction with its

replication origin, oriP, when provided in trans are claimed to boost protein ex-

pression plasmids. Furthermore, EBNA-1 appears to act as a transcriptional en-

hancer in human as well as rodent cells [78,81–84].

10.4.3

OPTIMIZATION OF TGE

Transient transfection needs to be optimized for each cell line and product. Once

the culture media is chosen, the variables to optimize are cell concentration at

the time of transfection, concentration of plasmids, and transfection reagent. The

current optimized protocol for VLP production in batch involves transfecting

the culture at 2 million cells/mL with a DNA concentration of 1 μg/mL, a

DNA:PEI ration of 1:2 [46].

10.4.4

ADDITIVES TO INCREASE TRANSIENT TRANSFECTION AND PROTEIN

PRODUCTION

Several additives have been tested to enhance transient transfection efficiency.

Lithium acetate and DMSO are used to increase cell membrane porosity to increase

the capacity of the polyplexes to enter the cell [85]. One of them is Nocodazole, that

acts in terms of cell cycle arrest in the G2/M phase, which is thought to enhance

nuclear uptake of the DNA/PEI complexes when the nuclear membrane dissolves

during mitosis [57].

Once the DNA is in the nucleus, another group of additives used to increase

production has also proven to be efficient in several cases. Sodium butyrate

[86–93], Valproic acid [90,94–97], and Trichostatin A [90,97,98] are used to inhibit

histone (HDAC), resulting in hyperacetylation of histones and, consequently, al-

terations in DNA transcription [86].

Hydroxyurea is used to block cell cycle in the G1 phase of the cell cycle, which

also leads to an increase in production [57,99,100].

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Bioprocessing of Viral Vaccines