With the revised EMEA position statement on Creutzfeldt-Jakob disease (CJD) and the safety of plasma and urine derived medicinal products1, there is an increased pressure on plasma product manufacturers to perform prion clearance studies.
Furthermore, with the trend in new vCJD cases occurring at a higher rate now in non-UK residents it is clear that a risk of potentially infected blood donors contributing to the plasma pool will continue for some time (Fig. 1). And, the EMEA guidance document on the investigation of manufacturing processes for plasma-derived medicinal products with respect to vCJD risk2 provides a framework assisting the design of prion clearance studies, and applies many of the principles that have served virus validation studies well over the years.
Investigations for prion removal or inactivation however still present challenges not encountered with virus studies. It is clear that for prions, we do not yet have the level of understanding of the nature of infectivity in blood that would allow such certainty of study design and interpretation. It is therefore not appropriate to use the term validation in the context of prion clearance studies.
In instances of vCJD implicated products, it is clear that any product manufactured from such plasma pools would be recalled in compliance with regulatory guidance. Recall events for vCJD, however, can also have significant consequences for subsequent batches of product manufactured using the same equipment. Such events highlight the importance of prion inactivation or clearance studies, and particularly the importance of demonstrating:
- Effective cleaning and sanitisation procedures to minimise the potential of vCJD carry over from previous batches.
- Effective prion removal by the manufacturing process, such that in the event of prion carry over, any risk to subsequently manufactured product can be considered small.
Solution studies investigating the inactivation of prion agents by a variety of procedures yield biphasic inactivation kinetics, with a rapid inactivation phase followed by a slower second phase.
A large proportion of the inactivation occurs during the initial rapid phase, and it was originally proposed that the slower second phase resulted from the presence of a subpopulation of prion agent more resistant to inactivation.
Additional studies have since demonstrated that qualities of the spike preparation and microenvironment of the agent can have a dramatic effect on the capacity of an inactivation procedure to inactivate prions.
Procedures which result in ‘fixing’ of the prion agent, such as drying, treatment with organic solvents or cross-linking with aldehyde based disinfectants results in an increase in the proportion of prion protein demonstrating resistance to inactivation (Taylor 1999). The fixing of the agent probably modulates resistance to inactivation through either preventing access to the inactivation solution (ie drying) or by preventing denaturation of the aggregated prion protein (ie aldehyde fixation).
The observation of increased resistance to inactivation following certain procedures has implications for the disinfection of biopharmaceutical equipment or surgical instruments suspected of CJD contamination.
Current practice for the GMP cleaning of biopharmaceutical equipment require that potential for carry over of product and contaminants must be minimised, that cleaning procedures are validated, and where components with an identified risk from TSEs are used, then prion cleaning/inactivation data may be required.
Sanitisation therefore becomes a significant topic of discussion for multi-product facilities, for slaughterhouses collecting raw components for subsequent manufacture and where re-dedication of equipment previously exposed to potential risk material is performed.
Given the critical nature of the microenvironment for prion inactivation, allowing equipment or instruments to dry, or treatment with organic solvents or fixing agents prior to disinfection is likely to increase the proportion of prion agent resistant to disinfection. Whilst prions will bind to surfaces, this is a property common to many proteins, and therefore prions should not be considered unique in this respect.
However, cleaning alone is insufficient to remove infectivity from surfaces exposed to significant prion contamination3. Significant protein deposits may increase the potential for ‘fixing’ of the prion agent, particularly if equipment has the opportunity to dry out. Cleaning procedures would be expected to reduce any protein load, and thereby improve the efficiency of any subsequent inactivation step. There are, however, few studies that have investigated this phenomena in sufficient detail.
In a recent study, bone surfaces devoid of any visible protein or fat deposits, yet containing still 6 logs of prion infectivity, when treated with 0.3M NaOH at ambient temperature resulted in complete inactivation of the remaining infectivity4. In this instance and in contrast to solution studies, no resistant prion sub-population was observed. Similar results have also been observed with steel wires exposed to prions and washed prior to exposure to inactivating agents3 and reinforces the importance of cleaning combined with decontamination.
The design and implementation of TSE clearance studies therefore requires careful planning and forethought, in order to ensure that the design fits with current regulatory guidelines, and furthermore that the data generated accurately represents the likely level of TSE inactivation.
Without experience these issues are not easy to resolve, and it is recommended to involve experts.
Dr Andy Bailey is CEO & Operations Director, ViruSure GmbH, Vienna, Austria. www.virusure.com
- Position Statement on Creutzfeldt-Jakob Disease and the Safety of Plasma- and Urine-Derived Medicinal Products. Committee for Proprietary Medicinal Products, 2004. EMEA/CPMP/BWP/2379/02 Rev. 1.
- Guidelines on the Investigation of Manufacturing Processes for Human Plasma-Derived Medicinal Products with Respect to vCJD Risk. Committee for Proprietary Medicinal Products, 2004. CPMP/BWP/CPMP/5136/03.
- Flechsig, E , et al , Transmission of scrapie by steel-surface-bound prions. Mol Med, 2001. 7(10): p. 679-84.
- Taylor, D M, Inactivation of TSE agents: safety of blood and blood-derived products. Transfus Clin Biol, 2003. 10(1): p. 23-5.