Introduction
Pharmaceutical manufacturing deals with selection and optimization of the cell source, media composition and physio-chemical bioreactor operating conditions to maximize the culture yield and productivity (Figure 1 ) 1-3. Escherichia coli(E. coli ) is a cost effective and attractive choice for producing therapeutic monoclonal antibodies, recombinant proteins and other biopharmaceuticals due to their rapid growth, minimal nutritional requirements, high product yield and transformation capability 4-11 With the millions of strains of bacteria, and gene-altering technology steadily improving, the possibilities are endless 12. One of the most recognizable products derived from genetically engineered E. Coli is the hormone insulin. Before being manufactured by bacteria, insulin was originally extracted from dogs and later pigs that was an extremely inefficient process, making the product rare and expensive 13. The advent of E. coli -produced insulin such as recombinant human insulin (Humulin) drastically increased its availability for diabetics 14. However, biopharmaceutical products manufactured using E. coli or other gram-negative bacteria are subject to endotoxin contamination 15-21.
Endotoxins are present in the outer cell wall of gram-negative bacteria that contribute to the organization and stability of the membrane 16-22. Endotoxin consists of three regions: a core polysaccharide, a long chain polysaccharide, and a non-polar lipid called Lipid A (Figure 2 ) 20,23. The core polysaccharide has an outer hexose region and an inner heptose region and the long chain polysaccharide is a strain-specific surface antigen (O-antigen) that consists of repeating oligosaccharide subunits 17,20. The core polysaccharide and the O-antigen are both hydrophilic while Lipid A is hydrophobic. The toxicity of endotoxin is associated with Lipid A 24-26. Lipid A triggers the production of pro inflammatory cytokines 27,28 and activation of the coagulation cascade 20,29 which can lead to sepsis and septic shock 30-34 . A pyrogenic reaction can be caused by as little as 1 ng of endotoxin per kilogram of body weight per hour 16-21,35. The standard unit for endotoxin measurements is an endotoxin unit (EU), which is equal to the activity of 0.1 ng of E. coli endotoxin 36,37. For intravenous applications, a maximum of 5 EU per kilogram of body weight can be administered to a patient per hour 36,38,39, but acceptable concentrations in biopharmaceutical products vary depending on the required dose 40,41.
Endotoxin is highly stable and is resistant to destruction by heat or pH 25,39,42. Additionally, endotoxins may form stable interactions with target therapeutic compounds that further complicates separations 23,43,44. Downstream processing of recombinant protein products accounts for approximately 45-92% of the total manufacturing costs 10,45,46. In addition to the downstream processing, the detection of endotoxins is absolutely critical for the safety of patients across the globe who rely on the purity of treatments prescribed 28. The purpose of this review is to discuss these aspects of an array of endotoxin detection and removal technologies.