In the pharmaceutical process, particularly for injectable drugs, removing bacterial endotoxins is crucial. Endotoxins, also known as lipopolysaccharides (LPS), are a key component of the cell wall of Gram-negative bacteria.
They often combines with other cellular substances, resulting in highly pyrogenic endotoxin complexes. These pose a significant risk to patients during intravenous or intramuscular injection.
Learning how to remove endotoxins is essential in the pharmaceutical field, as even trace amounts can cause severe adverse reactions and potentially threaten patients’ lives.
In this article, we will explore several common methods for endotoxin removal, including their advantages and disadvantages, to help you understand the technology and strategies for removing endotoxins.
Endotoxin risk in the pharmaceutical process
Endotoxins, derived from the outer membrane of Gram-negative bacteria, are lipopolysaccharides (LPS) released upon cell death or decomposition. LPS consists of bacterial-specific polysaccharides, non-specific core polysaccharides, and lipid A.
Lipid A is the main group of endotoxin’s various biological activities or toxic reactions. This group has no species specificity, so the lipid A structure of bacteria of various genera is similar, and its toxic reactions are similar, such as fever, hemodynamic changes, diffuse intravascular coagulation, and shock.
The O-specific polysaccharides determine bacterial species and type specificity as well as common antigenicity between different bacteria. Bacterial resistance to complement cleavage is also related to it.
Endotoxin, a lipopolysaccharide derived from Gram-negative bacteria, it is not a protein and has strong thermal stability. Even heating at 100℃ for 1h fails to destroy it, and wet heat at 115℃ for 30min only eliminates about 25% of its pyrogenic activity. Only extreme conditions like dry baking at 180℃ for 3-4h or 250℃ for 1-2h, or boiling with strong chemicals, can fully deactivate it.
Due to the hydrophobicity of lipid A, endotoxin tends to aggregate into high molecular weight polymers, making it difficult to dissolve in water. However, it is stabilized by attracting positively charged ions like Ca2+ and Mg2+.
LPS is prevalent in water-borne bacteria and a crucial component in parenteral products and equipment cleaning. An E. coli cell contains millions of LPS molecules, which are heat-stable and resist conventional sterilization. This necessitates separate testing for live cells and endotoxins.
Methods to remove endotoxins include dry heat treatment of glassware and rinsing for capping. In biotechnology, however, removing endotoxins bound to proteins is more complex and crucial.
How to remove endotoxins in the pharmaceutical process
Due to the significant harmfulness of endotoxins, FDA and other regulations mandate strict control of endotoxins to safe levels. While aseptic operation and cleanliness help control exogenous endotoxins, endogenous endotoxins in samples remain a challenge.
To reduce endotoxins in samples, maintain product properties, and improve safety, effective methods must be employed. Two key challenges are preserving product properties during removal and addressing low endotoxin concentrations, especially bound endotoxins. Enhancing endotoxin binding during protein concentration is a potential solution.
Endotoxin removal technology
In anion exchange chromatography, endotoxin is negatively charged at pH>2 and has a strong binding force with anion exchange media (such as Q or DEAE). In this way, the target protein can flow through the anion exchange filler, while the endotoxin is retained in the medium.
Another way is to allow the target protein and endotoxin to bind to the chromatography medium at the same time, elute the target protein first, and then wash out the endotoxin with a high salt buffer or NaOH.
In cation exchange chromatography, endotoxin is still negatively charged at pH 4.0 and cannot bind to the cation exchange filler, so It flows out with the mobile phase while the target protein binds, achieving endotoxin removal. thereby achieving the effect of removing endotoxin. In addition, surfactants (such as Triton X-114) can prevent endotoxin from binding to ion exchange media, thereby improving removal efficiency.
Membrane filtration removes endotoxins using positively charged membranes (such as membranes with Zeta potential). They separate negatively charged endotoxins via electrostatic adsorption, leveraging the toxin’s hydrophobic, hydrophilic, and charged areas. Endotoxins are common in nature, including water, air, and food, and originate from the outer cell wall of Gram-negative bacteria.
Our SANIendo® series filters combine the advantages of ion exchange and membrane filtration. They use a specially modified positively charged NY6,6 membrane surface to remove negatively charged endotoxins, viruses and cells in the fluid through electrostatic adsorption. This filter achieves a more efficient endotoxin removal effect through the combination of ion exchange and membrane filtration technology.
