operation are of incremental difficulty in implementation and operation. The fed-

batch mode adopts a special feeding strategy to extend the culture lifetime. The

addition of glucose, glutamine, and amino acids in a fed-batch bioreactor allows the

infections at higher cell densities. However, the development of an efficient feeding

strategy is still needed to enable virus replication at high cell densities. A rational

design of a feeding strategy relies on an extensive understanding of the metabolism

in cell culture and viral production phases. The perfusion mode usually results in

high product titers. In this mode, cells are retained inside the bioreactor using

different retention devices including acoustic filters, membrane units, centrifuges,

etc. For more details, refer to chapter 6 on process intensification. The nutrients are

fed and toxic by-products are removed continuously. After optimizing the perfusion

conditions like feed rate, infection time, and harvest time, a higher specific pro-

ductivity can be achieved when infecting the cells at a higher cell density [51,52].

11.2.2.4

Quantitation Methods of AdV Vector

The primary characterization of AdV are viral particle units (VPs) and infectious

viral particle units (IVPs). The most common method to physically determine the

total viral particles relies on the absorbance reading at 260 nm. A more precise

method to quantify the AdV is the anion-exchange–high-performance liquid

chromatography (AE–HPLC), in which the VPs were detected by photodiode array

detector at 260 nm [53]. For example, the AdV serotype 5 particles can be measured

by AE-HPLC method using a UNO Q column. The virus peak was eluted at

450 mM NaCl in about 8 min. Another quantification method for detecting the VPs

uses quantitative polymerase chain reaction (qPCR), which serves as a gold stan-

dard method. Compared with qPCR, the digital-droplet PCR (ddPCR) is more ro-

bust and has higher throughput since it provides absolute quantification with higher

sensitivity, omits the use of a standard, and requires less sample volume [54]. For

the quantification of IVP, the titers were usually determined by fifty-percent tissue

culture infective dose (TCID50) assay. The TCID50 quantifies the amount of virus

which can kill 50% of host cells or produce a cytopathic effect in 50% of cultured

cells [36,55]. For replication-competent AdV (RCA), determinations are generally

performed on pre-clinical and clinical studies. Results can be observed by cyto-

pathic effect on permissive cells such as A-549 and Hela cells after multiple di-

lutions. In addition, using PCR can enhance the sensitivity of the method. To

standardize the results of different quantitation methods and facilitate transfer of

pre-clinical and clinical data, a reference material for AdV vector has been gen-

erated [56]. For more details, refer to chapter 9.

11.2.3

EXAMPLES OF ADV-BASED VACCINES

11.2.3.1

Veterinary Vaccines

11.2.3.1.1

AdV-Based Rabies Vaccine

Control of rabies in wildlife remains an important challenge. The well-documented

rabies reservoir is bats. Raccoons, skunks, and foxes can be contaminated with rabies,

while domestic animals such as dogs can also be infected. In Canada, the rabies

Vectored vaccines

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