Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Table 1

Summary of endotoxin removal methods.

Method	Main features	References
Direct washing	with alkali ethanol, nitric acid and 70% ethanol in ultrasonic bath	[12, 15–18]
Microfiltration and ultrafiltration based on membrane adsorbers	convective mass transport and have shorter path length which translates to shorter residence time. Both filtrations show relatively good endotoxin clearance.	[3, 13, 27, 28]
Monolith	(i) PGMA has large pores which translate to rapid mass transfer, high adsorption kinetics and negligible low-flow resistance	[29, 31]
	(ii) Addition of Ethylenediaminetetraacetic Acid (EDTA) provides for the regeneration of metal-chelated particles with no morphology damage or protein adsorption capacity loss
Particle based adsorbents	Endotoxin and protein adsorption using DMAPAA takes place under a low salt condition	[13, 32, 33]
Two phase micellar system	(i) Involves separation of aqueous surfactant solution into micelle-rich and micelle-poor regions through excluded-volume interactions	[1]
	(ii) External agent notably Triton X-114 is needed to maintain the inherent biological activity of protein while reducing endotoxin level by 100-fold
	(i) Poly(𝜀-lysine) derived cellulose beads provide greater endotoxin selectivity
	(ii) High endotoxin retention is promoted using small pore size based on size-exclusion effects
Affinity chromatography	(iii) The high selectivity eliminates the need for multiple purification steps and reduces production costs	[5, 34–43]
	(iv) Reproducible, scalable and capable of specifically recognising and purifying super-coiled pDNA in arginine affinity chromatography
	(v) Arginine being non-immunogenic avoids interference with endotoxin assays
Immobilised metal affinity chromatography (IMAC)	(i) It can be applied for RNA, pDNA and endotoxin removal	[44, 45]
	(ii) There are situations involving special affinity interaction such as pure plasmid DNA binding specifically only to Fe3+ charged chelating compound
	(i) It uses composite polyacrylamide as the column which is highly porous
Size-exclusion chromatography (SEC) and ultrafiltration	(ii) Ultrafiltration is used if protein is not present. This method is capable of removing large endotoxin aggregate with alkanediol as one of the many agents used for effective endotoxin removal	[1, 34, 46, 47]
Anion-exchange chromatography (AEC)	It has rapid separation, wide selection of AEC media, sodium hydroxide (NaOH) sanitisation and does not require any solvents	[1, 6, 48]
	(i) Is thought to be more efficient than anionic exchanger in terms of endotoxin removal
Cation-exchange chromatography (CEC)	(ii) Polycationic ligands offer extremely strong attraction/binding for endotoxins	[1, 6, 13]
	(iii) PEI as a hydrophilic polymer has superior biocompatibility and exhibits hydrophobic interactions with endotoxin while PLL works well for protein recovery and still usable after binding capacity exhaustion
Immobilized hydrophobic ligands and reverse-phase chromatography (RPC)	Interact with non-polar protein surfaces through van der Waals forces for high endotoxin removal	[49, 50]