Design of Adsorptive Columns for Specific Pathogen Removal: Application to Staphylococcal Enterotoxin B Guangquan Wang † and Ruben G. Carbonell* Department of Chemical and Biomolecular Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Partner’s Building I, Suite 3200, Box 7006, Raleigh, North Carolina 27695-7006 The removal of pathogens such as toxins, viruses, bacteria, and prions in human blood, mammalian cell culture media, fermentation broths, food items, and water streams has gained increasing importance in ensuring product safety and in combatting acts of terrorism. Adsorption processes can play an important role in removing such pathogens from solution without affecting other desirable components. Adsorptive columns that can remove specific families of pathogens would need to achieve a reduction of several logs in pathogen concentration. This requirement is much more stringent than the normal yield requirements associated with adsorptive separations aimed at product recovery and purification in a process stream. This paper considers the design of an adsorptive column aimed at reducing the concentration of infectious agents from a known volume of solution by several logs in a fixed amount of time. The general rate (GR) model of chromatography is used in the analysis, including all major transport and kinetic steps in the adsorption process. The theory, with no adjustable parameters, is shown to predict with great accuracy the effect of residence time on the log removal of staphylococcal enterotoxin B (SEB) from solution using an affinity resin with a small peptide (YYWLHH) that has been found to bind specifically to this toxin. Introduction Many biological products derived from either human or animal origins contain an inherent risk of pathogen contamina- tion. Minimizing the risk of transmission of infectious diseases is of primary importance during production. Protein therapeutics derived from human plasma may be contaminated by a host of bacteria and viruses, such as human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human parvovirus B19 (1). In addition, an unrealized but potential threat to the safety of plasma products is the presence of transmissible spongiform encephalopathies (TSEs), such as bovine spongiform encepha- lopathy (BSE or mad cow disease) in cattle and Creutzfeldt- Jakob disease (CJD) and variant CJD (vCJD) in humans, which are associated with the pathogenic and infectious prion protein (1-3). Bacterial endotoxins derived from the outer membranes of Gram-negative bacteria are some of the main contaminants in the manufacturing of recombinant DNA products that use these bacteria as host cells (4). Dairy foods such as milk are potentially contaminated by a bacterium, Staphylococcus aureus, which releases staphylococcal enterotoxins that are leading causes in food poisoning (5). To prevent pathogen transmission, safety considerations require careful screening of raw materials as well as virus validation studies through all steps in the process. Pathogen levels can be reduced by either inactivation or removal (2, 3). Common inactivation methods to denature pathogens such as viruses involve the use of heat, chemicals, fatty acids, and low pH (2). The main drawback of pathogen inactivation is that it can also result in losses of product activity. It has also been found that many inactivation strategies are inefficient to reduce the levels of nonenveloped viruses (2). Removal strategies can have the advantage of reducing pathogens while maintaining the activity of biological products. It has been shown that membrane filtration, ion-exchange chromatography, and pre- cipitation can result in significant levels of adventitious pathogen removal in biopharmaceutical processes (1, 6). Even though several logs of removal of viruses, prions, and other pathogens have been demonstrated as a result of a sequence of protein purification steps, changes in process variables can have a significant impact on this adventitious pathogen reduction. Adsorption columns, such as ion exchange, hydrophobic interaction, and affinity columns, are widely used in purification processes for biotherapeutics, and at the same time, they often remove pathogens from the process stream. Regulatory con- straints on the clearance of pathogens are typically expressed in terms of the potential reduction in risk of infection, but ultimately the reduction in risk is related to logs of pathogen removal from the process. In the particular case of removal of pathogens by adsorption from a biological mixture, design requirements dictate the removal of at least 4 or 5 logs of infectious agent for product safety (7). In some special cases, it might be advantageous to use specific ligands on adsorptive resins that can recognize a particular class of pathogen. For example, ligands might be designed so they recognize a highly conserved region of a class of toxin or a bacterial or viral protein. A good example of this would be the use of a prion removal column to reduce the risk of prion infection from purified biotherapeutics. Other examples arise from potential hazards to the water and food supply from bioterrorism. Adsorptive columns may play a role as a final * To whom correspondence should be addressed. Tel: 919-515-5118. Fax: 919-515-5831. Email: ruben@ncsu.edu. † Present address: Talecris Biotherapeutics, Process Development & Technology Department, 8368 US 70 West, P.O. Box 507, Clayton, NC 27520. 1358 Biotechnol. Prog. 2006, 22, 1358-1367 10.1021/bp060126l CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/12/2006