![]() ![]() Incubation at room temperature to promote association with lipid A is followed by refrigeration that causes the detergent to gelatinize, facilitating its removal with still-associated endotoxin.Īll these methods leave the largest and most exposed part of the endotoxin molecule unexploited: the O-antigen, also known as the O-polysaccharide. The only method that effectively exploits hydrophobicity of lipid A at present involves addition of the Triton X114 surfactant ( 17). The high salt conditions required for protein binding copromote formation of stable self-associations among endotoxins that make the lipid A region inaccessible. Hydrophobic-interaction chromatography might be expected to target the hydrophobicity of the O-and N-linked fatty acids of the lipid A region, but it has not proven competitive. IMAC is more complex due to the necessity of selecting and binding metals in addition to the usual tasks of method development, but it also tolerates high salt concentrations. Proteins that elute from HA in chloride salts at low phosphate concentration support the best endotoxin removal. They also bind iron, aluminum, copper, and zinc ions, among other metal ions ( 16). Endotoxin phosphoryl residues have especially high affinity for the calcium component of hydroxyapatite ( 15). The metal affinity of the core polysaccharide and lipid A phosphoryl groups can be exploited with either hydroxyapatite (HA) or immobilized metal affinity chromatography (IMAC). But it also shares their burdens: elevated expense and intolerance of rigorous sanitization conditions. It bears the same conveniences of other biological ligands: exquisite selectivity, no need for sample equilibration, and minimal development burden. Increasing conductivity or reducing pH to enable antibody flow-through tends to compromise endotoxin binding in parallel.īiological affinity chromatography with a recombinant version of the protein that enables the Limulus amoebocyte lysate (LAL) test also exploits the core polysaccharide and lipid A regions ( 14) of endotoxins. Endotoxins bind well under such conditions. These limitations have their least impact with alkaline proteins such as IgG1 monoclonal antibodies that flow through columns at mildly alkaline pH and low conductivity. In many cases, multimodals support more effective endotoxin removal than traditional anion exchangers, but they have two limitations: They bind many proteins, and their effectivity is dramatically altered by variations in pH and conductivity. These methods focus on the negatively charged oxygen atoms associated with the carboxyl and phosphoryl residues of the core polysaccharide and lipid A regions of the molecule (Figure 1). Examples include traditional anion exchangers and a number of multimodal electropositive adsorbents such as histidine, polymixin B, and Capto Adhere and Toyopearl NH2-750F media ( 9– 13). Positively charged chromatography media historically have dominated the field. Figure 1: Endotoxin basic structure and chemical potentialsĮndotoxin removal methods typically focus on particular regions of an endotoxin molecule.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |