Agilent Technologies Fractionation Application Note

Fractionation of Fungal Fermentation
Broth using Solid-Phase Extraction

Application Note

Bioactive Pharmaceuticals, Agrochemicals

Authors

Liam Evans and Jonathan Steele
Hypha Discovery Ltd

Abstract

This application note describes a very effi cient clean-up and fractionation method
using a polymer-based, solid-phase extraction (SPE) product, Bond Elut Plexa.

This quick and effective method shows how unwanted sources of assay
interferences, such as proteins and oligosaccharides, can be removed allowing the
fractionation of the smaller bioactive pharmaceutical and agrochemical molecules
of interest from tropical higher fungi into more precise groups of polarity.

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Introduction

Molecules from nature have an
excellent proven track record of
providing initial leads for development
into new pharmaceutical and
agrochemical products. To discover
new bioactive pharmaceutical and
agrochemical molecules from tropical
higher fungi, a new method that
stimulates fermentation of fungi has
been developed. The huge increase in
chemical production is shown in the
chromatographic analysis (Figure 1).

The composed fermentation products
vary greatly in molecule size and
polarity and require an effective
fractionation prior to identifi cation
and bioassay. For example, the
macromolecules have to be removed
before the compounds of interest can
be selectively collected according
to their polarities. Preparative
chromatography is one technique,
which can be used to clean-up and
fractionate. However, the high price
and the long analysis time make this
technique ineffi cient for our work.

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Figure 1. Chromatogram showing comparison of compound production using Hypha fermentation and
conventional fermentation. Hypha’s fermentation provides increased titres and expression of new
molecules

LOAD
Water-rich, hydrophilic
surface allows excellent
phase transfer of analytes into
the polymer core.

WASH
Analytes that have crossed
the hydrophilic layers will
remain tightly bound in the
hydrophobic core.

Mi nutes

Hypha Fermentation

Conventional
Fermentation

ELUTE
Specially engineered pore
structure allows excellent
mass transfer out of the
polymer.

A very effi cient method is the clean-up
and fractionation on a new polymer­based, solid phase extraction (SPE)
product, Bond Elut Plexa. The primary
extracts generated from the proprietary
method are processed through the
SPE cartridges to remove proteins,
enzymes, oligosaccharides and other
biopolymers, which are known to cause
interference in target-based screening.

Analyte

Hydrophobic

Pores

Large endogenous proteins do
not bind to the surface of the
polymer and cannot access
pore structure

Figure 2. The water-wettable, hydroxylated exterior allows excellent fl ow of bio-fl uid samples. A
gradient of polarity on the polymer surface shunts small analytes to the more hydrophobic center
of the polymer bead where they are retained. As the surface is highly polar and entirely amide-free,
binding of proteins on the polymer surface is minimized, resulting in cleaner samples and reduced ion
suppression

core

Interferences washed away
without leaching the analytes
of interest

Clean extract with high
recovery

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