ABSTRACT

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders that includes scrapie and bovine spongiform encephalopathy (BSE) in animals, and Creutzfeldt-Jakob disease (CJD) in humans. The prion agent shows remarkable resistance to common physiochemical inactivation procedures and conventional chemical disinfectants. Methods that reduce TSE infectivity, such as treatment with a strong base or autoclaving, are not always compatible with maintaining the biologic activity of proteins. Therefore, purification processes that separate therapeutic proteins from agents that might cause TSE infectivity are necessary to reduce the possibility of transmission. It is reasonable to assume that available methods to remove viruses and bacteria during the manufacture of gonadotropins are also capable of removing prions. In this study, the authors explored whether a nanofiltration process used during the extraction process of follicle-stimulating hormone from human urine can be effectively leveraged for removal of prions under conditions used for the manufacture of urine-derived gonadotropins.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders that includes scrapie and bovine spongiform encephalopathy (BSE) in animals, and Creutzfeldt–Jakob disease (CJD) in humans (1). The agent responsible for such infections is a protein molecule called PrP^Sc, a conformational variant of the normal host protein, PrP^C. The key event in the pathogenesis of TSEs is believed to be the conversion of PrP^C into the pathogenic isoform, PrP^Sc (2). The infectious agent causing TSEs is the prion, which may be identical to PrP^Sc, a partially protease-resistant isoform of a membrane glycoprotein termed PrP^C (3).

While growing evidence suggests that infectivity can be transmitted in blood, the presence of PrP^Sc in urine and associated infectivity remains to be fully confirmed. However, the presence of the noninfective PrP^C in some urinary gonadotropin drugs has been recently reported. This finding was not totally unexpected because of the ubiquity and relative abundance of this physiological protein in various regions of the body (9). Indeed, some papers have reported the presence of the infective PrP^Sc in the urine of animals in models of disease (10).

For these reasons, the debate regarding the use of urinary gonadotropins and their potential risk of transmission of variant CJD is still ongoing (11). Risk assessments need to be conducted in both blood-derived products and urine-derived products, such as gonadotropins, to determine the actual likelihood of transmitting infection and assess actions for risk mitigation.

The prion agent has several unusual characteristics, including a remarkable resistance to physiochemical inactivation procedures such as heat, ionization, ultraviolet light, microwaves, and irradiation, and to conventional chemical disinfectants such as detergents, alcohol, glutaraldehyde, and formalin (12). Methods that reduce TSE infectivity, such as treatment with a strong base or autoclaving, are not in most cases compatible with maintaining the biologic activity of proteins (13). Therefore, purification processes that separate therapeutic proteins from TSE infectivity are necessary to reduce the possibility of TSE transmission by biological components.

Several techniques remove or inactivate PrP^Sc throughout the manufacturing processes and are applicable to the production of human menopausal gonadotropins and recombinant follicle stimulating hormone (rFSH). Stringent regulatory measuresare already in place to account for viruses and bacteria during the manufacture of gonadotropins, and it is reasonable to assume that such steps can remove significant amounts, if not all, of PrP^Sc during the manufacturing process. Trials to remove TSE pathogens from biological materials have been made by several investigators using precipitation, chromatography, and filtration (14–22).

The virus filtration step evaluated in this study is used by the Institut Biochimique SA (IBSA) during the process of extracting FSH from human urine. FSH from human urine undergoes a purification process including precipitation with solvents, chromatography, and ultrafiltration. These steps lead to a process that efficiently minimizes the risk of viral contamination in the final product through inactivation, partition, and removal of the potential pathogenic agents.

This study evaluates the prion removal or inactivation achieved by nanofitration membranes (Viresolve normal flow parvovirus [NFP], EMD Millipore) when used according to IBSA's process conditions for the purification of human-derived gonadotropins.