size, shape, and surface structure. Additionally, physicochemical features like

isoelectric point (pI), surface hydrophobicity, and the presence of viral envelope can

play important roles in the design of the DSP train. Given this biodiversity, there are

no universal purification processes for viral vaccines. However, there are common

steps and unit operations providing a good starting point, as depicted in Figure 7.3.

Furthermore, achieving the high levels of purity required for viral-product clinical

application requires a complex cascade of unit operations, that go further than the

traditional purification methods discussed before. The purification scheme usually

includes a unit operation for clarification, intermediate purification (capture and

concentration), and polishing of the product of interest. However, the number and

order of the unit operations might vary based on the virus of interest as well as final

therapeutic dose and usage, given the fact that the impurities specifications are

based on the dose and final use.

In the following sections, each unit operation will be described and the tech-

nology trends in vaccine manufacturing will be mentioned.

7.2.1

HARVEST AND CLARIFICATION

When the virus or viral vector is harvested from the cell culture, at the end of the

upstream processing (USP), the resulting harvest material contains, apart from the

viral product of interest, processing reagents (media components), cell debris, and

host cell-related molecules (host cell proteins, DNA). All these contaminants present

a health risk if present in a final formulation of a vaccine candidate. So, there is a need

for their removal to a certain extent before that virus preparation is used as a bio-

therapeutic, specifications being set by regulatory authorities. The time of harvest

(TOH), as defined in Chapter 6 as the upstream process development–process in-

tensification, should be established, taking into consideration different factors like

product quality, process reproducibility, virus stability, and not only the process

productivity [15]. Harvesting later in the upstream cell culture process will lead to

reduced cell viabilities and consequently to an increase in host-cell contaminants i.e.,

host cell protein (HCP), host cell DNA (HCDNA), and cell debris. This might

strongly affect to following efficiency of the purification process.

Viruses used for vaccination are generally produced by cell infection, and their

release is dependent upon the virus cycle since they can be found intra- or extra-

cellularly. In the case of lytic viruses assembled intra-cellularly, a cell lysis step is

necessary to release the neosynthetized particles as for adenovirus or adeno-associated

virus. Such cell lysis or cell disruption is thus performed prior or after clarification step.

It can be performed by mechanical (homogenization, sonication) or chemical (freeze-

thaw cycles, detergent addition) methods [16]. Considering adenovirus manufacturing,

at a laboratory scale, the purification process consists of a cell lysis step using freeze

and thaw methodologies, followed by density gradient ultracentrifugation and a final

desalting step [17,18]. This process is successful at a small scale, achieving high purity

level while maintaining a low total to infectious viral particle ratio (below 30).

However, this methodology has strong limitations associated with the processing time

and scalability. This is why cell lysis at a larger scale is commonly performed by the

addition of detergents [19]. One detergent widely used is a mild non-ionic detergent

Downstream processing

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