Process Analytical Tools for the Monitoring and Characterization of Bacterial Toxins

Abstract: In the era of proteomics, glycomics and ultimately metabolomics, with constantly increasing knowledge regarding the human biosystem, the number of specialized therapeutic agents which will be developed against different diseases is predicted to be on the upswing. An increasing portion of these new drugs will, according to recent predictions (Labrou, 2003), be of proteinaceous origin produced according to biotechnological standards. Hence, down stream processes (DSP) in which large amounts of specific target proteins are to be processed and purified, with high demands on process monitoring and control, will be of further increasing importance in the future. In the light of these predictions, the U.S. Food and Drug Administration (FDA) has proposed a set of new initiatives that primarily address the development of process analytical technology (PAT) as a concept within pharmaceutical manufacturing. The idea with PAT is to stimulate creative strategies for improved process developments, which in the end should lead to increased productivity concomitantly with the assurance of acceptable end-product quality (U.S. FDA, 2002). The definition of PAT involves the use of an overall process optimisation strategy applied at all production levels. Hence, the utilization of advanced bioprocess monitoring tools, which could lead to reduced production cycle times and prevention of rejects, scrap, and the need for re-processing, is covered in the description. A recent tendency within bioproduction monitoring is the shift in focus from the target molecule itself towards an increased control of the impurities present (World Health Organization, 1999). Among the relevant contaminants typically present in a bioproduction process, bacterial toxins have received high priority from the regulatory agencies (Ecker et al., 2005). In fact, a clear analytical strategy for the control of e.g. bacterial endotoxins is a necessity for the recombinant production of any FDA-approved pharmaceutically active proteinaceous agent (U.S. FDA, 1985). Currently, the only available method for endotoxin detection is the limulus amebocyte lysate (LAL) assay which is based on the Limulus lectin derived from the American horseshoe crab,. The LAL assay is expensive and time-consuming. Moreover, it is non-functional for the detection of endotoxins in trace amounts. Hence, the development of new, less labour-intensive methods with high sensitivity and specificity for the direct detection of endotoxins is highly desirable. Another important category of contaminants associated with biotechnological production comprises the exotoxins. This class of bacterial toxins typically includes soluble proteinaceous compounds, continually released by living bacteria. Extreme toxicities together with a typical ability to retain high activity under harsh conditions and in dilute solutions make exotoxins exceptionally competent as poisons, often with high mortality rates. The treatment of toxic by-products in biotechnological production is strictly regulated and the strategies for the removal of toxins associated with the product are often complex and completely dependant on the product application. A parallel situation is the handling of waste liquids remaining from the process. Fermentation of cell strains, capable of excreting exotoxic compounds to the surroundings, will consequently give waste liquids that require detoxification before being discarded. Hence, cost-effective methods for performing such tasks could be of interest to the manufacturer as e.g. the cost of sending the material for incineration could be a heavy toll on the profit margins. The objective in this thesis work was to investigate different means to analyze, characterize and process selected toxins of bacterial origin related to biotechnological production. With a vast array of compounds falling into the category of bacterial toxins, two different types were chosen; staphylococcal enterotoxin B (SEB), a 23 kDa exotoxin produced by Staphylococcus aureus and the heterogeneous group of endotoxins derived from the outer cell-wall of the gram-negative bacteria Escherichia coli.