New PCR Test Rules Out BSE

12/02/2004

Celsus Laboratories (Cincinnati), a manufacturer of heparin and other anticoagulant and antithrombotic agents, has developed a new test that will authenticate the porcine origin of its critical raw material and rule out bovine adulteration at what is expected to be < 1 animal-per-lot detection level.

The company had already developed screening methods to determine the presence of prion antigens (Pharmaceutical Manufacturing, July/August, p. 9) and validate its processes. A new test, based on a polymerase chain reaction (PCR) method, will allow the company to quantify porcine- and bovine-specific genes in crude heparin lots. Celsus will use the test as a criterion for supplier and raw material approval, says Alan Cardin, Ph.D., vice president of research and regulatory affairs. At this point, the company is not licensing the process, but may do so in the future.

Livestock traceability of animal-derived products is becoming a key issue in gaining drug and device approvals, Cardin says, primarily because of the risk of exposure to the transmissable spongiform encephalopathies (TSE), most notably bovine spongiform encephalopathy (BSE) otherwise known as “mad cow disease.”

To date, regulators have focused on country of origin to manage BSE risks, Cardin says, taking into account each country’s historical incidence of BSE, animal species based on animal susceptibility to infection, and tissue categories based on considerations of infectivity load.

Regulatory agencies are now placing greater emphasis on records documenting animal traceability as a compliance requirement of raw material integrity. This approach, however, is burdensome on the drug manufacturer, it is neither practical nor reliable, Cardin says. It is particularly unreliable where pooling of raw materials is required, he explains.

For example, although hogs are not susceptible to TSEs, porcine material may be adulterated with bovine material. A single batch of Heparin Sodium may be derived from the intestinal mucosa from over 50,000 hogs and the pooling process may involve multiple change-of-custody points in the supply chain between different businesses, i.e. rural farms, slaughter houses, mucosal collection and crude heparin extraction, each representing a point of introduction for contamination by susceptible species. Thus, absolute assurance over the integrity of each and every batch of product with respect to both the species origin and health status of every animal in the pool is not possible based only on the suppliers’ certification and periodic audits by the manufacturer.

Species-specific identity testing by real time polymerase chain reaction (PCR) of animal-derived raw materials could definitively verify the animal species origin of the crude starting materials provided that suitable DNA extraction methods, species-specific target genes and high fidelity amplification procedures can be devised, Cardin says. Because of the known sensitivity, selectivity and target specificity of PCR, its use may be suitable for identification tests and detection of species adulteration. Such a test could be used as a criterion for supplier approval as well as for crude and finished batch certification.