using iodixanol (OptiPrep™) density gradient was also established for the retrovirus

purification. This method requires fewer manipulations, when compared with other

density gradient mediums, and leads to higher titers and lower processing times due to

the lower viscosity of iodixanol. Given the lack of toxicity of this compound, there is

no need for its removal [12].

Although high-speed preparative centrifugation has been used for the past dec-

ades for virus purification, its use presents several drawbacks. Indeed, it is related to

high initial investment costs, limited scalability, and expensive maintenance, being

suitable for laboratory scale to obtain high concentrated virus preparations [13,14].

Additionally, viruses may suffer a loss of infectivity during the long period required

to fractionate the gradients, commonly between 16 and 90 hours.

Although ultracentrifugation was progressively replaced by new operations for

manufacturing applications in the last two decades, it is still the method of choice for

new vaccine candidates. Indeed, for such cases, partial knowledge of their physico-

chemical properties, especially for early clinical trials, ultracentrifugation is still the

safer purification approach. For that reason, single-use ultracentrifuges were developed

while single-use manufacturing processes were deployed in the industries, like for

example Ksep® (Sartorius Stedim) and CARR UniFuge® (Pneumatic Scale Angelus).

Both equipments have the advantage to apply low shear on cells, enabling the harvest of

intact cells as product or discard them as a by-product. This is especially important for

budding enveloped viruses, where a cell lysis step is usually not required.

However, the trend in the industry is trying to avoid the centrifugation processes,

and increasingly exploring the chromatographic and membrane-based purification

techniques.

7.2

PURIFICATION STRATEGIES FOR VIRUSES

The purification of viruses is not a simple task given their biological complexity

compared with other biotherapeutics, like antibodies (mAbs). Indeed, they differ in

FIGURE 7.2 Density gradient ultracentrifugation using caesium chloride to purify adeno-

virus and AAVs.

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Bioprocessing of Viral Vaccines