Potency testing of influenza vaccines and batch release

Single radial immunodiffusion (SRD)

The SRD test for inactivated influenza vaccines was developed at NIBSC in the 1970s. This assay measures the quantity of haemagglutinin (HA) antigen present in a sample – such as a vaccine – using an immunological reaction between antibodies and the test antigen. 

It was recommended for use in batch release of influenza vaccines by the World Health Organisation (WHO) in 1978 and remains the gold standard method for the potency determination of inactivated influenza vaccines.

Reagents for the SRD test

NIBSC has been generating the necessary reagents for the SRD test for decades. Two types of reagents are required for the SRD test:

• an antiserum reagent
• a calibrated reference antigen reagent

Antiserum reagent

The antiserum reagent is prepared – usually in sheep – against purified HA protein of a virus representative of the strain(s) recommended by WHO for inclusion in the vaccine.

So for a trivalent vaccine three antiserum reagents are needed – one for an A(H1N1) virus, one for A(H3N2) and one for the influenza B virus component of the vaccine. 

Antigen reference reagent

The antigen reference reagent is made from inactivated purified whole virus preparations. It is calibrated against a primary standard. Assigning a value for HA content of the primary standard is done by physico-chemical methods. The primary standard serves as calibrator for the secondary antigen reference reagent. This is produced in large quantities and is freeze-dried for long-term stability.

We perform the calibration of the antigen reference reagents in collaboration with a small network of laboratories – called the WHO Essential Regulatory Laboratories (ERLs) – which are at NIBSC, the US Food and Drug Administration (FDA), the Australian Therapeutic Goods Administration (TGA) and the National Institute of Infectious Diseases (NIID) in Japan.

The calibration process follows a protocol agreed between the four ERLs and endorsed by the WHO’s Expert Committee on Biological Standardisation (ECBS).

The influenza reagents we produce are available to manufacturers and independent control laboratories – regulatory laboratories – worldwide. The NIBSC catalogue lists all influenza reagents that are currently available.

Experimental method for the calibration of primary liquid standards

In collaboration with Jun Wheeler’s group at NIBSC, we have introduced an experimental method for the calibration of primary liquid standards. We are evaluating this method – isotope dilution mass spectrometry (IDMS), which was originally described by researchers at the US Centers for Disease Control and Prevention (CDC) – alongside the conventional calibration methodology for primary standards.

We collaborate with US CDC and the Center for Biologics Evaluation and Research (CBER)/FDA (USA) in this evaluation. The new method may replace or complement our current procedures in the future.

Batch release

As the UK’s National Control Laboratory, the NIBSC carries out independent batch release testing of influenza vaccines.

In recent years interest has grown in the development of alternative potency assays. We have been active in this area by:

• taking part in key meetings and workshops relevant to the topic of potency testing of influenza vaccines
• performing research on new potency assays
• evaluating potential alternative potency assays

The advent of quadrivalent influenza vaccines that contain four strains – two influenza A strains of subtypes A(H1N1) and A(H3N2) as well as two influenza B viruses of the two lineages co-circulating in humans (Victoria and Yamagata lineage) – has brought new challenges to potency testing.

The SRD test is subtype- and type-specific. This means that any of the three components of a trivalent vaccine can be tested in the presence of the other two components without interference.

However the two influenza B virus components are immunologically cross-reactive to varying degrees, so there is occasional interference. We currently working to address this issue, for instance by generating antiserum reagents with minimal cross-reactivity.