preferred for crude cell supernatant rich in high biomass content, due to their higher

capacity to retain impurities within the filter. Their mode of operation is not only

based on size exclusion, but also hydrophobic and electrostatic interactions. Some

filter aids can be added to depth filters, such as diatomaceous earth (DE), that

revealed to improve the filter efficiency in retaining particles [26]. Depth filters play

mainly with two factors for particle retention: size exclusion and adsorption. Some

filters, such as Sartopure® PP3, stand out given their very low unspecific binding.

This allows retaining most of cells and cells debris [27].

On the other hand, microfiltration membranes using TFF retain only impurities

larger than the pore size and have a lower impurity holding capacity. They are,

therefore, suitable for a secondary clarification step. Both kinds of clarification filters

(depth filters and membrane devices) are suitable for scale-up in vaccine manu-

facturing and both have already been incorporated in the manufacturing processes of

viral vaccines [28–30]. Generally, clarification is a complex step with a number of

available technologies. As presented in Table 7.1, there is no universal solution since

the selection of the filters and operation parameters requires critical handling.

As discussed previously, it is of importance to wisely select the TOH, as, it can

strongly impact clarification step. Indeed, harvesting cell cultures with low cell

viability increase the presence of cell debris in the extracellular medium thus re-

ducing filter capacity and further blockage. To assess the clarification process ef-

ficiency, solution turbidy monitoring is an important parameter. It also enables the

detection of the filter capacity which is related to the fouling of the membrane [38].

Membrane fouling is commonly the consequence of the formation of a polarized

layer on the filter surface due to impurities’ accumulation present in the cell su-

pernatant. Operating membrane microfiltration through tangential flow filtration

(TFF) allows avoiding such formation of the polarized layer since the cross-flow

decreases the formation of a “cake” on the membrane surface.

Membrane devices such as hollow fibers or cassettes can be used for the clar-

ification step using TFF. However, the latest upstream technologies advances

(Chapter 5), especially the high cell densities processes, are challenging such fil-

tration operations. In the last decade, several virus’s production systems have been

described to produce at cell densities above 10⁷ cell/ml, namely PER.C6 cell line

grown up to 10⁸ cell/mL for HIV vaccine candidate [39]; MDCK cells infected with

the influenza virus at 5 × 10⁷ cell/ml [40] and, more recently, CR.pIX cells reaching

2.5 × 10⁷ cells/mL for modified vaccinia Ankara production [41]. All of these

productions employed an advanced alternating tangential flow (ATF) perfusion

system, using a hollow-fiber device. Another filtration technology widely applied to

high-cell densities is body feed filtration (BFF). Here few filter aids are added to the

crude bulk, such as diatomaceous earth (DE), to enhance the filter capacity [42].

Another novel technology of Repligen for fed-batch clarification is TFDF™, which

combines benefits of tangential flow (TF) and depth filtration (DF) in a single-use

system with pore size ranging from 2 to 5 μm. This system was successfully applied

to separate lentiviral vectors from cell debris in batch and perfusion production

modes [43].

During vaccine manufacturing, there is an extra concern with host-cell DNA

removal depending on a risk assessment to evaluate possible side effects with the

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Bioprocessing of Viral Vaccines