of calculating probabilities of virus detection, based on factors such as signal in-

tensity, coverage across multiple targets within the same virus, and signals from

closely related viruses.

4.4.1.2.3

Mass Spectrometry

Protein-mass spectrometry by MALDI TOF is also considered as an attractive al-

ternative for the detection of viruses, but its application is very limited by the

sensitivity required and by the complexity of the samples typically used. Coupled

separation techniques like 2-D gel electrophoresis, in addition to peptide analysis by

LC-MS/MS, are powerful methods used for detection of unknown viruses in

complex samples.

These new methods using mass-spectrometry still need to be validated for their

use in the detection of adventitious viruses.

4.4.2

CELL LINE−BASED ASSAYS AND THE FUTURE

4.4.2.1

Potency Tests

The efficacy of a vaccine is generally demonstrated by the ability of the immune

system to protect against disease. The potency is a measurable characteristic of a

vaccine that was correlated with the protection in the clinical study.

In practice, potency is used to demonstrate consistency (discriminating potent

and subpotent) between vaccine lots, on the assumption that that the accepted range

of potency correlates with the safety and the efficacy of the product. This range is

part of the submission file approved by the regulatory authorities.

A broad panel of methods is currently used for potency tests and is dependent on

the properties of the product and other analytical considerations.

4.4.2.1.1

In Vivo Potency

The most direct nonclinical method for testing the efficacy of a vaccine is a chal-

lenge test in an animal model (typically rodents). The objective is to demonstrate

the prevention of mortality or pathogenesis in vaccinated animals.

If a challenge model is not available or feasible, the immune response to the

vaccine is measured. A proxy for efficacy is evaluated by the detection of antibody

titers, either by neutralizing activity or by ELISA, or by hemagglutination assays. In

vivo potency tests for vaccine release are mainly required on specific inactivated-

virus vaccines (Polio Salk) or purified products including antigen combinations.

Because animal-based tests are becoming ethically unacceptable, as well as

being expensive, time-consuming, and often inaccurate, these tests are tending to be

superseded by in vitro potency tests [30].

4.4.2.1.2

In Vitro Potency

The selection of the in vitro test is primarily shaped by the mechanism of action of

the vaccine.

The in vitro potency tests for recombinant or purified vaccine are often im-

munoassays (e.g., single radial immunodiffusion potency test for the influenza

vaccine and the enzyme-linked immunosorbent assay for recombinant Hepatitis B

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Bioprocessing of Viral Vaccines