affect the transfection efficiency [59,61–64]. DNA and PEI chain length, PEI charge

density, structure and chemical modifications, the nitrogen/phosphate (N/P) ratio, the

DNA and PEI concentrations, or the complex preparation and addition process have

also a major impact on the TGE efficiency [43,44,55,64,65]. Furthermore, the cell

line, cell culture medium composition, cell concentration, time of contact of DNA/

PEI complexes with cells, or the addition or replacement of medium in the trans-

fection protocol have also a remarkable influence on the in vitro results [43,61,66].

Design of experiments (DoE) have been implemented to optimize some of these

multiple variables in a faster and rational way [41,48,67,68]. On top of that, the idea of

DNA/PEI complexes as nanoparticles has brought new parameters, such as size,

particle concentration, or morphology, that might also play a role in the delivery

process [69–73]. Apart from the myriad of variables, a large heterogeneity in the

DNA/PEI polyplex population itself has been described [70].

10.4

METHODS TO IMPROVE THE PRODUCTION PROCESS

In the Gag-VLPs production process different elements can be optimized. One

efficient way to optimize the relevant aspects in the process can be design of ex-

periments (DoE). By using this methodology, the different variables can be opti-

mized at the same time studying their interactions and the overall contributions to

the result. The classical approach where several variables need to be optimized

performs a primary screening experiment to determine which variables have a

positive effect on the result. These screening can be performed using the Plackett-

Burmann design of experiments. After identifying the variables with a positive

effect, the next step is to find the optimal levels for each one of them using surface

response designs. This can be done using central composite design (CCD) and,

when the number of variables to optimize increases, the Box-Behnken approach.

The result of these experiments are statistically significant data that are used to find

a mathematical model and an optimal combination of the different variables.

Finally, the determined optimal conditions must be validated experimentally.

10.4.1

SERUM-FREE MEDIA

Culture media has a high impact on the maximum cell density reached, duration of

the production phase and final product concentration.

The need of serum removal from bioprocess was recognized decades ago. First

attempts in serum-free media (SFM) development included components of animal

origin mimicking the components supplied by the serum, such as insulin, transferrin

and lipids of animal origin as well as other poorly defined mixtures (extracts, hy-

drolysates). Currently, the overall trend in SFM formulation is to suppress com-

pletely animal-derived components to avoid any potential contact with new viruses

or prion strains. The increasing number of animal-derived component-free (ADCF)

and chemically defined (CD) media formulations available in the market and the

relatively recent commercial availability of recombinant versions of key serum

proteins produced in Escherichia coli or yeast (e.g., albumin and transferrin), as

Recombinant vaccines: Gag-based VLPs

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