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.
TSEs are neurological diseases caused by a proteinaceous agent called a prion. These diseases include Creutzfeldt–Jakob disease
(CJD) in humans and scrapie in sheep. The disease-causing protein is thought to be the result of alternate folding of a protein
containing the same amino acid sequence found in the circulation of uninfected individuals. How the normal protein is converted
to the infective form is the subject of much speculation and experimentation. One particular TSE - BSE, or "mad cow disease"
- is of particular concern for producers of biological products.
Table 1. Scrapie agent load over alkaline treatment time course.
Previously, TSEs were thought to be species-specific. That is, the disease in one organism could not be transmitted to another.
However, events in the United Kingdom and Europe have indicated that a disease called variant CJD (vCJD) is quite likely derived
from BSE (1). The route of transmission is thought to be the ingestion of neural tissue from a diseased animal. The BSE agent
has now been shown to be transmissible to sheep by direct intravenous injection (2).
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
- the source animals and their geographical origin
- the nature of the animal material used in manufacture, and any procedures in place to avoid cross-contamination with higher-risk
- the production processes and the quality assurance system in place to ensure product consistency and traceability.
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).
Prion Clearance Studies
Similar to viral clearance studies, the objective of TSE clearance studies is to assess manufacturing process steps that might
be considered effective in inactivating or removing TSE agents and making quantitative evaluations of the level of reduction.
TSE clearance studies are designed like conventional viral clearance studies (7). The nature of the spiking material and the
endpoint assay, however, are unique to TSE clearance studies.
Figure 1. Photomicrographs of brain tissue of golden Syrian hamster; (a) control injected intracerebrally with saline; (b)
scrapie lesions are seen in a brain infected intracerebrally with the hamster-adapted 263K scrapie strain.
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.