Sponsors of therapeutic biologicals must demonstrate they are not introducing disease-causing agents into patients. There is a perceived risk - among the public and some regulators - of spongiform encephalopathy transmission in biotechnological products using bovine-derived cell culture components. Although there have been no incidents of transmission to humans of spongiform encephalopathies, particularly bovine spongiform encephalopathy (BSE), precautions must be taken. By employing animal-sourced materials from younger animals, which have never shown effects of the disease; using a robust process to produce cell culture supplements; and purifying the therapeutic products, the perceived risk is reduced.
However, when compared with other adventitious agents, prions - which cause transmissible spongiform encephalopathies (TSEs) - are a greater concern because they are so difficult to inactivate, and there is currently no rapid, reliable technique to detect their presence. In this article, we present information about prion diseases, critical elements of TSE agent clearance studies, and a case study for inactivation of TSE by use of alkaline conditions during the manufacturing process of a cell culture media supplement.
These findings indicate that concern about the use of bovine materials in the production of biopharmaceuticals is valid. However, according to European, World Health Organization (WHO), and U.S. Department of Agriculture (USDA) regulations, serum and plasma derived from veterinary-screened animals in non-BSE countries are considered low-risk materials (3–5). A risk assessment for vaccines produced using bovine-derived material is provided by FDA on its website (6).
As stated in the Note for Guidance on Minimising the Risk of Transmitting Animal Spongiform Encephalopathies via Human and Veterinary Medicinal Products from the Center for Proprietary Medicinal Products (CPMP) (5), the risk of TSE contamination can be reduced by addressing
Unfortunately, BSE is appearing in areas of the world once thought to be free of this prion agent, and we clearly do not know where it will be found in the future. The CPMP Note for Guidance states: "Manufacturers should continue their investigations into removal and inactivation methods to identify steps/processes, which would have benefit in assuring the removal or inactivation of TSE agents" (5).
Hamster strain 263K. With relatively short incubation periods of less than 12 months and the highest titer of any of the TSE rodent adapted strains, the hamster 263K strain is often chosen as a model for TSEs in validation studies. This strain is well characterized and the incubation period and brain histopathology are well documented (Figure 1). A recent study supports the validity of hamster scrapie as a model for other TSE agents (8). The method of preparing the spiking material is an important consideration in the design of a clearance study. The use of a crude brain homogenate, as the spiking material in an inactivation step, provides the greatest challenge, whereas a less viscous preparation has been found superior for some removal steps (9).
The resistance of the hamster-adapted 263K strain to inactivation is similar to that of other TSE agents, and methods that have been shown to successfully inactivate prions have been reported (10). Sodium hydroxide has been reported to be an effective inactivating agent for TSEs, and it is widely used in biotechnology to sanitize processing equipment (5,11).
Inactivation of Prion Agent We used EX-CYTE growth enhancement media supplement (Serologicals Corporation) as our case study for inactivating a prion agent. This medium is a water-soluble mixture of lipoproteins derived directly from bovine serum or plasma. It is used as a supplement to reduce the use of fetal bovine serum in cell culture media and as a component in serum-free cell culture media to promote the growth of mammalian cells and the production of genetically engineered proteins. Our case study involves the validation of the alkaline inactivation step for EX-CYTE.