known as Triton X-100. It solubilizes the cell membranes allowing the viruses release.

Several other applications of Triton X-100, commonly formulated at 0.1% exist, as the

protein solubilization, the viral sub-unit preparation, and the enveloped virus in-

activation [20,21]. However, there is evidence that Triton X-100 can have undesirable

effects on the environment due to endocrine disruption properties during its degrada-

tion. Thus, it was added to the REACH (Registration, Evaluation, Authorisation and

Restrictions of Chemicals) list, forbidding its use from 4 January 2021, forcing the

companies to work on eco-friendly substitutes. One alternative recently reported was

Polysorbate 20, which is a stable, non-toxic, and non-ionic surfactant widely used in

domestic and pharmaceutical applications [22]. In this work, the efficacy of Polysorbate

20 was evaluated and compared with Triton X-100. Results showed no negative effects

on the adenovirus’s purification train and an increased virus recovery and impurities’

removal. Other alternatives, such as sodium deoxycholate for the AAVs’ purification

[23] and CHAPS for adenovirus’s purification [24] have already been applied.

Nevertheless, detergents added to the culture should be removed, as they can have an

impact on the next downstream operation. The increase in virus yield thus obtained

should compensate for the extra efforts to achieve the required purification level for the

viral vaccine.

After cell lysis, there is an increase in the impurities level (host cell proteins,

host cell DNA, and cell debris) needing to be removed by centrifugation or

filtration technologies such as depth filtration or microfiltration. Although clar-

ification is not always categorized as a downstream operation and is sometimes

neglected in vaccine manufacturing, this unit operation is of high importance in

virus purification. In fact, an optimized and efficient clarification step can

strongly impact the overall purification process’ costs and also reduce the man-

ufacturing footprint, by reducing the subsequent size of chromatography columns

and buffers’ consumption.

Given the biological diversity of vaccine types, several series of operations are

required to achieve an efficient clarification [25]. The first operation aims to remove

larger particles, like remaining cells and cell debris usually applying centrifugation.

Afterwards, a second step is used to remove low molecular weight particles thanks

to filtration techniques. Centrifugation is frequently used as the method of choice

for clarification of cell culture production-based products at large scales as it can be

operated both in batch or continuous mode. However, several drawbacks of this

technique makes of filtration techniques new methods of choice.

First, the cost of investment for large-scale centrifugation for equipment is high

compared to filtration techniques and their sanitization procedures are critical and

laborious. Then, even though centrifugation is possible to operate at large scales, the

limited sample capacity of the centrifuge is still one of its main disadvantages.

Thus, given the improvement in upstream processing technology, enabling higher

titers, filtration techniques have gained interest in vaccine clarification.

Filtration techniques used in clarification through membrane microfiltration can

be performed either by normal flow filtration (NFF, also known as dead-end fil-

tration) or tangential flow filtration (TFF, also known as cross-flow filtration).

For viral-based products, filtration membranes have pore sizes in the range of

0.1–10 µm. Depth filtration is normally used in dead-end mode, and is usually

Downstream processing

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