Lipids are naturally-occurring hydrocarbons2 that are poorly soluble in water and soluble in non-polar solvents such as ether and chloroform. They serve many important functions in cells and other biological systems. For example, lipids serve as structural components in cellular membranes, provide energy reserves (e.g. triglycerides), and are vital in the role of metabolism as vitamins and coenzymes.
However important lipids are to living systems, they can interfere with analytical assays and biopharmaceutical production. For example, the presence of fatty acids in multi-residue pesticide analysis in food products can cause ion-enhancement during MS analysis. Phospholipids can lead to ion-suppression during preclinical and clinical studies during pharmaceutical development. Removal of endotoxins, lipopolysaccharides derived from the cell membrane of gramnegative bacteria (e.g. E. coli), is critical in biopharmaceutical applications involving gene therapy products and transfection studies.
In this report we discuss the use of a new synthetic calcium silicate hydrate called LRA (Lipid Removal Agent) for the selective removal of lipids, macromolecules containing lipid moieties and other biological macromolecules in both analytical and process scale applications.
LRA (Lipid Removal Agent) media is a synthetic calcium silicate hydrate developed for the selective removal of lipids in the production of biopharmaceuticals. LRA media is highly specific for lipids, lipopolysaccharides (endotoxins) and lipoproteins in the presence of other biological macromolecules (e.g. albumin, immuno globulins, and other proteins). LRA is often used for the removal of lipids in biotech process streams and analytical assays that require lipid removal during sample preparation. Please see Table 1 for detailed specifications of LRA and Table 2 for LRA’s lipid removal capacity.
In this application, bulk LRA was incubated with cryoreduced source plasma at increasing LRA concentration levels for a period of 3 hours at ambient temperature. Following incubation, the sample was centrifuged and the supernatant was analyzed via nephelometry for removal of protein. Table 3 shows that proteins, for the most part, are not removed by LRA. Although some non-specific adsorption occurred for plasma ceruloplasmin, IgM and alpha-2 HS glycoprotein occurred at 80 g LRA / L plasma, adsorption to apolipoprotein A1 and B was specific relative to the majority of the other plasma proteins endogenous to the sample.
In this study we demonstrate LRA’s high capacity for phospholipids. LRA (40 mg) was packed into a 3 mL SPE tube between two 20 μm PE frits. A 0.5 mL aliquot of a 0.1 mg/mL phosphatidylcholine in 80% acetonitrile was loaded onto the LRA SPE tube with the aid of an SPE vacuum manifold. The resulting eluate was analyzed for phosphatidylcholine breakthrough via LC-UV analysis. The LRA adsorbent offered 91.4% removal of the phosphat idylcholine with a single pass through the LRA SPE tube (data not shown).
Endotoxin removal is of critical concern in the development and manufacture of biopharmaceutical products. Zhang and coworkers1 demonstrated that LRA media has an effective endotoxin removal capacity of 6 x 106 endotoxin units (EU) per gram LRA, and offered rapid adsorption kinetics as well. For example, over 99.9% endotoxin in a 5000 EU/mL sample was adsorbed onto the LRA media after mixing for only 2 minutes. They further demonstrated the performance of the LRA by comparing it against a number of other commercially-available endotoxin removal agents.
Although LRA was developed to selectively remove lipids, researchers have exploited LRA’s selectivity for other purification purposes. Winters and coworkers2 characterized the adsorption mechanism of LRA and have developed a cost effective and simple method for removing key impurities from E. coli lysates in the purification of supercoiled plasmid products. In a single adsorptive contact step, they were able to remove genomic DNA and open-circular plasmid whereas supercoiled plasmid remains unbound. When combined with LRA’s selective affinity towards detergents and endotoxins, LRA media offers a powerful purification tool in downstream polishing during bioprocess purification.
LRA media is a synthetic calcium hydrate developed for the selective removal of lipids in the presence of other biological macromolecules. Its high capacity and adsorptive properties are ideal for bioprocessing purification, but it can be scaled down for analytical sample prep purposes as well. In this report we showed LRA’s capacity and adsorptive efficiency across a range of lipids including endotoxins, phospholipids and lipoproteins. LRA’s unique selectively can also be extended to other applications as well including the purification of supercoiled plasmid DNA.
Although LRA is currently available in bulk packing, LRA media can also be supplied in common LPLC hardware such as SPE tubes, VersaFlash® cartridges, Büchner Funnels, etc.
Customers using LRA media in cGMP production of biopharmaceuticals can request an LRA Regulatory Support Package through Supelco Technical Service. The support package contains detailed information regarding LRA manufacturing and quality control procedures/specifications.