Index: Volume 2 Number 1
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Lipid contaminants often pose a problem in the processing of biologically derived solutions. These contaminants typically cannot be removed by conventional centrifugation or filtration. Historically, lipids have been extracted using solvents such as ether and chloroform, or solubilized with surfactants. In most cases, these methodologies are either impractical or cause protein denaturation. Solid-phase methods such as the addition of fumed silica have also been employed, but these methods tend to adsorb proteins, making them unattractive.

Cleanascite™, is a solid-phase reagent designed specifically for the selective removal of lipids, floating fats, cell debris, and mucinous impurities from biological systems. This reagent effectively adsorbs lipids but does not cross-react to any significant extent with antibodies (both lgG & lgM), glycoproteins or nucleic acids. Cleanascite's™ cross-reactivity with other types of proteins has been shown to be minimal with only small losses of protein in some applications. At application ratios typically in the range of 1 volume of Cleanascite™ to 2-4 volumes of sample, no dilution is observed. When used for clarification of ascites fluid the supernatant can be applied directly to an affinity or ion-exchange column, precluding the need for ammonium sulfate precipitation. Cleanascite™ facilitates easier subsequent purification and can increase the life of membrane and chromatographic apparatus, thus providing economic benefits. Cleanascite™ is supplied as a suspension in saline and has no buffering capacity. The following study illustrates the high clarifying capacity and high recovery of antibodies and proteins using this reagent.

Optimization of Lipid Removal and Protein Recovery

Cleanascite™ was added at a volume relative to the degree of lipid contamination in biological samples. To establish appropriate ratios, a 2% phospholipid mixture was formed by homogenizing saturated and unsaturated fatty acids and cholesterol. The turbidity of this mixture was designed to be similar to that in ascitic fluid (determined by A₅₅₀). This mixture was spiked with 10 mg/ml of purified mouse antibodies. Increasing amounts of Cleanascite™ were added to each sample and mixed for 10 minutes at RT using an end-over-end mixer. The samples were centrifuged at 2000 x g for 15 minutes to pellet the lipid-bound matrix. The lipid removal efficiency was measured by turbidity (A₅₅₀) and the recovery of antibodies was determined by protein colorimetric assay (Pierce BCA reagent) (Figure 1).

Figure 1:
Optimization of Cleanascite™ to sample ratio. Lipid removal from phospholipid-IgG Antibody recovery from phospholipid-IgG Antibody recovery from human serum

An analysis of Cleanascite™ ratios was also performed on human serum. Antibody recoveries were determined by an outside contractor (Figure 1). Additional testing was performed on various client samples. The results are illustrated in Table 1. In all studies, the recovery of the desired compound exceeded 90%.

Table 1: Client Samples

Practical Applications

Cleanascite™ has been demonstrated to enhance many applications including:

• Initial clarification of ascites fluid in the production of monoclonal antibodies.

• Removal of lipid haze from bacterial and yeast homogenates in the purification of recombinant proteins.

• Clarification of biological extracts such as serum, tissue culture and organ homogenates during the isolation of nucleic acids.

• Removal of surfactants from biological extracts in the processing of integral cell membrane components, i.e., proteoglycans and antibodies.

• Elimination of milk fats in the purification of proteins from milk such as in processing from transgenic animals.

• Removal of lipids from whole blood or sera, beneficial in the processing of blood products such as hemoglobin.

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