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.