required [17]. More than one unit operation is sometimes used to achieve a desired

clarified product. The first clarification aims to remove larger specimens, like cells,

while a second one will reduce colloids and other sub-micron particles [111].

Conventional unit operations such as sedimentation or flocculation are still being

used in vaccine development. These methods are simple to design and operate, but

do not remove cellular debris or sub-micron particles [111,112]. Normal flow fil-

tration (NFF, also known as dead-end filtration) or tangential flow filtration (TFF)

are also used in large-scale production processes. Nonetheless, process optimization

and low flow rates are sometimes reported due to mechanical stress or titer loss

caused by virus adsorption. Depth filters, which contain filter aid, enhance retention

of cell debris and contribute to improve NFF results, while the use of hollow fibers

or membrane cassettes with high cutoffs, reduces the shear stress of viral particles

improving TFF. In addition, membrane-based approaches might benefit from the

use of inert materials such as regenerated cellulose (RC), polysulfone (PS), poly-

ethersulfone (PES), and polyvinylidene fluoride (PVDF) [113,114].

One or more concentration steps are typically performed after clarification.

Affinity, ion-exchange (IEX), or hydrophobic interaction chromatography (HIC) with

different matrices, such as polymer-grafted beads, monolith, membrane adsorbers, or

gigapore resins have been successfully applied to VLPs and viral vectors [115–121].

Alternative methods such as two-phase extraction or flocculation have been also

described [121,122]. TFF was successfully reported for influenza VLPs, where

Carvalho et al. described a total recovery of 76% in a complete membrane-based

purification process [123,124]. In addition, a nuclease treatment is often added to

reduce dsDNA, especially when anion IEX chromatography is applied [17].

In order to polish VLPs from process-related impurities and prepare them for

formulation, diafiltration or size exclusion chromatography (SEC) are mainly used

[121]. Polishing steps aim to remove dsDNA, HCP and other contaminant particles

that might co-elute with VLPs, such as EVs, adventitious viruses or BEVS. While the

quantification and removal of the first two has been widely described, the separation

of VLPs from other contaminant particles is highly difficult due to their similar

physicochemical properties. Moleirinho et al. recently reported the development of an

affinity chromatography method to separate VLPs from BV [125], whereas Reiter and

co-workers achieved a reduction on BEVS content by polymer-grafted chromato-

graphy [126]. Regarding EVs, no separation method as such has been described.

Limitations at analytical level for the specific quantification of VLPs and EVs present

in the same sample may strongly contribute to this fact. Concretely, the available

analytical tools do not allow the direct differentiation between VLPs and EVs due to

their same origin, composition and physicochemical properties. On the other hand, the

enrichment of HIV-1 Gag VLPs over total particles has been reported by TFF, IEX

chromatography and heparin affinity chromatography [119,124,127].

Removal of adventitious agents must be demonstrated according to the proce-

dures established by regulatory authorities. Common methods to remove or in-

activate viruses are UV or gamma-irradiation, sterile filtration, detergent treatments,

or high-temperature incubation, which may compromise the candidate integrity or

biological activity. Mitigation of such risk should start at the selection of raw

material level. The use of animal-component free, chemically defined cell culture

Recombinant vaccines: Gag-based VLPs

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