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March 4, 2013 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 13-05 

TO: Biologics Licensees, Permittees, and Applicants

 Directors, Center for Veterinary Biologics

 Veterinary Services Leadership Team

 

FROM: Richard E. Hill, Jr. /s/ Richard E. Hill, Jr.

 Director

 Center for Veterinary Biologics

SUBJECT: Use of Polymerase Chain Reaction (PCR) Assays to Measure Potency of 

 Inactivated Protein-Based Biologicals

I. PURPOSE

This Notice clarifies Center for Veterinary Biologics (CVB) policy regarding the use of 

PCR assays in the potency evaluation of inactivated protein-based biologicals. 

II. BACKGROUND

All serials of biological products must be tested for potency before their release for 

marketing. Most such serial release testing is done with assays that measure potency, 

although some utilize laboratory models based on efficacy surrogates or correlates, such 

as laboratory animal vaccination-challenge models or host species serology. Potency is a 

quantitative measure of a preparation’s active ingredient. For vaccines, the active 

ingredient is the protective epitope. (This is a simplification that is useful for discussion, 

but we recognize that vaccine activity may be a more complex interplay between more 

than one epitope and other components such as adjuvants.) By providing a quantitative 

measure of the active component, an ideal potency assay has a direct relationship with 

efficacy. 

Historically, most in vitro assays used to test the potency of inactivated protein-based 

biologics have been designed to quantify antigen. The target antigen is selected for its 

role in the protective immune response; preferably it is detected (measured) via an agentneutralizing monoclonal antibody. This, combined with strict manufacturing controls, is 

intended to mitigate the risk associated with quantifying one representative antigen of a 

complex microbial agent in an in vitro platform. 

United States 

Department of 

Agriculture

Animal and Plant 

Health Inspection 

Service

Veterinary Services

Center for Veterinary 

Biologics

1920 Dayton Avenue 

PO Box 844 

Ames, IA 50010 

(515) 337- 6100
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 13-05 

Page 2 

Recently we have received several proposals to use PCR to test inactivated protein-based 

biologicals. PCR assays measure the presence of genes that code for proteinaceous 

antigens in vaccines. PCR assays do not provide assurance an antigen has actually been 

produced by a gene or that the antigen has retained its immunogenic form through 

inactivation and other downstream product processing. Many antigens, especially 

bacterial, are conditionally expressed, based on nutrient availability and/or environmental 

stressors. If unfavorable growth conditions down-regulate the expression of a critical 

antigen, this would not be detected at the nucleic acid level. Thus, PCR requires critical 

assumptions beyond those required for an antigen-quantification assay, and the CVB 

maintains that these assumptions represent excessive risk unless mitigating factors can be 

implemented. 

One potential mitigating factor is to combine PCR with another assay(s) which, when 

considered together, provide superior assurance of serial potency than either assay alone. 

To date, the most frequent reason for considering PCR for potency testing is because a 

product contains multiple closely related microbial strains, and strain-specific reagents 

are not readily available to develop antigen-based assays that will not cross-react with 

other fractions in the product. In this case, the CVB will consider a cross-reacting 

protein-based assay to measure overall cross-reacting antigen content in conjunction with 

PCR to assess the probable relative input by each strain. 

III. POLICY 

When PCR is proposed to test the potency of an inactivated protein-based biological, it 

shall be in the context of a multi-component potency assessment. One component of the 

assessment must characterize protein antigen in the completed product. 

IV. IMPLEMENTATION/ APPLICABILITY

This Notice simply clarifies existing CVB policy regarding the use of PCR in potency 

testing inactivated protein-based biologicals. No changes are being implemented.