SuperFluids™ CXF

In SuperFluids™ CXF fractionation of marine microorganisms, fermentation pellets are first contacted by SuperFluids™ for microbial cell disruption (as shown by the disrupted yeast cell) and secondly for the polarity-guided fractional extraction of extracellular and intracellular bioactive constituents. The SuperFluids™ critical fluid fractionation (CXF) process produces six fractions or more of increasing polarity. Butanol and aqueous fractions are also prepared, for a total of eight or more fractions per fermentation broth or thirty-two or more fractions per marine microorganism based on four fermentations of each microorganism with different combinations of carbon and nitrogen designed to increase biodiversity.

In the discovery of complex natural molecules, pure compounds will provide the clearest benefit in bioactive screens. It is, however, prohibitively expensive to isolate and purify new natural product molecules without a pre-defined biological activity. Additionally, most potent biologically active compounds often occur in trace amounts that are likely to be missed in any broad-scale purification of natural products from a complex matrix. SuperFluids™ CXF fractionation produces partially purified compounds in a rapid fashion that can be automated.

The SuperFluids™ CXF process, for first cellular disruption and secondly, polarity-guided fractional extraction of biomass such as marine microorganisms and terrestrial plants enhances the drug discovery process. Our research has resulted in a much higher proportion of bioactive “hits” in certain screens from these SuperFluids™ fractions, at lower than normal metabolite concentrations. The SuperFluids™ CXF process produces partially purified extracts and enhances the probability of “clean” hits.

In comparative screening programs with the Bristol-Myers Squibb Company (BMS), research determined that the hit rate from over 40 screens was higher in SuperFluids™ fractions than in either butanol or aqueous fractions; compare a hit rate of 2% with 1.1% and 0.6%, respectively for butanol and aqueous supernatants [BMS tested each of 8,320 fractions in 40 different screens for a total of 332,800 tests].

A blinded study was conducted with Pfizer Research Laboratories, Groton, CT to examine the ability of SuperFluids™ to extract a different set of metabolites, as compared to conventional extraction techniques. SuperFluids™ fractions had more unique peaks as compared to butanol extracts. Since the solvating properties of SuperFluids™ differ from those of conventional organic solvents, a different spectrum of metabolites was extracted.

These studies have demonstrated that SuperFluids™ CXF technology produces a unique spectrum of secondary metabolites, reduces interference from nuisance compounds, minimizes background noise in sensitive molecular assays and enhances the probability of "clean" hits.

The SuperFluids™ CXF technology (process and apparatus) has been automated for high throughput sample preparation of natural products such as marine microorganisms and medicinal plants. This fractionation process is readily implemented in parallel units to further increase the speed of sample preparation for antiviral and anticancer screening.

The process can be readily adapted to large-scale manufacturing by Aphios’ SuperFluids™ CXP manufacturing technologies that are both scalable and cost-effective for complex natural product molecules.

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