format, showed quantification results and coefficients of variation similar to those

obtained by SRID, and a quantification range between 1–10 μgHA/mL. Durous, et al.

[64] demonstrated the use of an antibody-independent SPR assay for HA quantifica-

tion, employing fetuin containing α-2,3 and α-2,6-linked terminal sialic acid as ligands.

The assay, shown to be specific only to active trimeric HA, displayed a large dynamic

range (between 0.03–20 μgHA/mL) and negligible non-specific interactions with

different culture medias or MDCK by-products.

9.6

DOWNSTREAM PROCESSING OF INFLUENZA VACCINES

The development of new processes for influenza vaccine production raises new

challenges for downstream processing. Depending on the vaccine type (IIV, LAIV,

RV) and production substrate (eggs, cell culture), the required steps for product

purification, as well as the techniques to be employed, may differ [65]. The pur-

ification process starts with the harvest of virus-containing media, which is done by

extracting the allantoic fluid in egg-based systems, or by collecting supernatant/cell

pellet after centrifugation for cell substrates. An additional step of extraction with

detergent, performed on the collected cell pellet, is required for RVs produced using

the baculovirus system in insect cells. Clarification is then performed either through

a centrifugation or a filtration step, depending on the production platform. A virus

inactivation step is required for cell and egg-based IIVs, which is usually done

chemically by the addition of formaldehyde or β-propiolactone [65]. After that,

purification steps vary largely for different types of vaccines.

For IIVs produced in eggs, following steps include concentration and purifica-

tion by zonal centrifugation, virus disruption performed by centrifugation in the

presence of cetyltrimethylammonium bromide CTAB, followed by polishing, and

vaccine formulation. Flucelvax (Seqirus), a sub-unit IIV produced in MDCK cells,

follows a different process: concentration and initial purification is done through a

chromatographic step. Host DNA is then removed by benzonase treatment and virus

disruption is performed by centrifugation with CTAB, followed by an ultra-

centrifugation polishing step and vaccine formulation [66,67]. A different process is

employed for Flublok (Sanofi-Pasteur), a RV composed of recombinant HA. After

clarification, a capture step is performed using an ion-exchange chromatography

resin, followed by another chromatographic step (hydrophobic resin) for further

removal of contaminants. Host cell DNA is removed by Q membrane filtration

followed by a final ultrafiltration step and vaccine formulation [68].

9.7

CONCLUSION

In the last decades, a robust system for influenza surveillance and vaccine pro-

duction was developed, interconnecting the World Health Organization, national

centers, regulatory agencies, and manufacturers around the globe. Other fast-

mutating infectious agents, such as viruses from the coronavirus family and notably

the SARS-CoV-2, responsible for the COVID-19 pandemic, could benefit from

similar systems to reduce the severity of outbreaks and increase responsiveness to

eventual pandemics. The development of new viral vaccine technologies, such as

Manufacturing of influenza vaccines

233