requiring specific quantification methods. Antibody-based techniques, including the

SRID assay—the only assay formally approved by the WHO and other regulatory

bodies for influenza quantification for vaccine formulation – measure the biologi-

cally active trimeric form of the HA protein. Since these techniques are time

consuming and depend on the generation of specific antibodies, they are mainly

employed for vaccine formulation [53]. While faster methods for influenza quan-

tification are described, such as flow-virometry [54], RP-HPLC [55], and ion ex-

change HPLC [56], various challenges still hamper their use for routine influenza

quantification [53]. The hemagglutination assay, that measures HA content based

on the reticulation of red blood cells, is one of the most common assays for influenza

quantification. Although it is a fast and inexpensive alternative for routine quantifi-

cation during the production stage, it presents high variations based on the quality of

cells and expertise of the operator, resulting in increased errors that restrict its use for

process development. A decrease in HA assay errors was demonstrated by Kalbfuss,

et al. [57] when an automated read-out for plates and a shift in dilution factor were

introduced. The neuraminidase assay, based on the measure of the NA activity, has

shown to be a fast and accurate assay for influenza quantification that could be applied

during the production process of some types of vaccines (before virus inactivation).

However, increased cost of reagents and high background noise depending on sample

composition have shown to be major problems for assay application [57]. Finally,

infectivity measures such as the TCID50 assay, quantify total infectious particles based

on the ability of the viral suspension to kill host cells [58]. While extremely time

consuming and labor intensive, these methods are crucial for viral stock preparation.

The development of fast and cost-effective methods for influenza quantification could

have a great impact over the development of upstream processes. New methods, such as

the ddPCR for total viral particles assessments [59,60] and the surface plasmon re-

sonance for HA quantification [61–64] are being developed, the latter being especially

interesting given the possibility of its application for online measurements during

production [64]. Nilsson, et al. [62] developed an antibody-dependent SPR assay for

the quantification of HA in process samples. The assay, developed in an inhibition

TABLE 9.4

DNA, mRNA, and viral vectored influenza vaccines currently in clinical trials

Company

Vaccine type

Phase

Vaccitech

Viral vectored – Modified Vaccinia Ankara (MVA)

Phase II

Vaxart

Viral vectored − Adenovirus

Phase II

Altimmune

Viral vectored − Adenovirus

Phase II

AlphaVax

Viral vectored − Alphavirus

Phase II

Moderna

mRNA

Phase II

Inovio

DNA

Phase I

Mymetics

Virosomes

Phase I

Source: clinicaltrials.gov, Accessed on: September 2021 [ 52].

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