Recombinant biopharmaceuticals are manufactured using complex biological systems, such as bacteria, yeast, baculoviruses or
mammalian cells. However, regulating consistent yields, monitoring contamination and validating product efficacy during the
manufacturing process are areas of concern and represent major challenges for biopharmaceutical manufacturers.
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High batch rejection rates of biopharmaceuticals, which are common during production, can usually be attributed to contamination
by biological agents such as microorganisms or byproducts produced by these microorganisms, including toxins or pyrogens.
Endotoxin contamination of biopharmaceuticals is a serious issue and may infringe on workforce safety. Apart from adherence
to FDA regulations and strict international quality standards, many US pharmaceutical companies must also deal with the Occupational
Health and Safety Administration, which ensures that contamination control is much more than just a housekeeping activity.
In Europe, this task is taken up by the European Agency for Safety and Health at Work.
Bacterial endotoxin is another term used for lipopolysaccharides (LPS), complexes that are located in the outer cell membrane
of Gram-negative bacteria and blue-green algae.1 Gram-negative bacteria are widely used in biopharm manufacturing to produce recombinant DNA products, such as therapeutic
proteins. LPS subunits are complex amphiphilic molecules with a molecular weight (MW) of approximately 10–20 kDa2,3 and vary widely in chemical composition both between and among bacterial species. LPS complexes tend to aggregate and form
large structures that have an average MW > 10 kDa.
LPS is a potential cause of pyrogenic reactions in parenteral drug products because these complexes can act as a strong immunostimulant
that activates the complement system by the alternative (properdin) pathway4 upon entry into the human blood circulation. This can cause death of the individual who is administered with the drug.5,6
A pyrogenic reaction can be caused by only a small amount of endotoxin — approximately 0.1 ng/kg of body weight. The standard
reporting unit for endotoxin data is one endotoxin unit (EU) — the equivalent of 0.1 ng. A typical gram-negative bacterium
contains 10–15 g of LPS, which means that at least 105 bacterial cells are required to contribute 0.1 ng of endotoxin.
The chemical structure of endotoxin
Figure 1: Lipopolysaccharide (LPS) structure and location in the bacterial membrane.
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LPS consists of three components or regions: Lipid A, an R-polysaccharide and an O-polysaccharide (Figure 1).
Somatic (O) antigen or O-polysaccharide is attached to the core polysaccharide and consists of repeating oligosaccharide subunits
made up of three to five sugars. The O-polysaccharide maintains the hydrophilic domain of the LPS molecule and also contains
the major antigenic determinant (antibody-combining site) of the gram-negative cell wall.
Toxicity of endotoxin has been found to be associated with Lipid A, whilst immunogenicity is associated with the polysaccharide
components.
