## Production of brefeldin-A

TG McCloud^1^, MP Burns^1,\ 2^, FD Majadly^1^, GM Muschik^1^, DA
Miller^3^, KK Poole^3^, JM Roach^3^, JT Ross^3^, and WB Lebherz, III^3^

^1^*Chemical Synthesis and Analysis Laboratory,* ^3^*Fermentation
Production Facility, Program Resources, Inc/DynCorp, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
Maryland 21702-1201, USA*

**Fermentation conditions are described for the production of the
antitumor antibiotic 7-(S)-brefeldin-A (brefeldin-A) in liquid culture
by *Eupenicillium brefeldianum*, (B. Dodge) Stolk and Scott, ATCC 58665.
An analytical hplc method was developed which allowed rapid quantitation
of the compound during fermentation. A kilogram of brefeldin-A was
isolated from a fermentation at the 6800-liter scale.**

**Keywords:** brefeldin-A; *Eupenicillium brefeldianum*; antitumor
antibiotic

### Introduction

Brefeldin-A, (synonyms: ascotoxin, cyanein,decumbin, lunatin) a
13-carbon macrolide lactone antibiotic first iso-

lated by Singleton in 1958 from *Penicillium decumbens* \[25\], has been
subsequently identified as a metabolite from

*P. brefeldianum* \[10,27\], *P. cyaneum* \[4\], and other sources
\[1,26,30\] (Figure 1). The structure was determined by Sigg \[24,32\].
The 7-(R) epimer has been reported from a different microbial source
\[8\]. Biosynthesis of brefeldin-A has been investigated by Hutchinson
and others \[6,12,13,29,31\]. Several total syntheses have been reported
\[2,5,9,11,14,18\]. Because of unexceptional antibiotic properties,
interest in the compound was restricted to bio-medical research
laboratories until recently. The discovery of a previously unknown
mechanism of action on animal cells, the disruption of intracellular
protein transport by dis-

sociation of endoplasmic reticulum following treatment

with brefeldin-A, \[21,22\], and selectivity for certain cell

types of the 60 human tumor cell lines which constitute the

anticancer screen used by the National Cancer Institute

(NCI) to detect potential new chemotherapeutic agents \[20\]

has revived interest in the compound. Because of these findings
brefeldin-A was selected by the Developmental Therapeutics Program
(DTP), NCI, for scaled-up production to provide sufficient quantities
for further *in* *vitro* and *in vivo* testing. Other biological effects
of brefeldin-A have recently been reviewed \[3,16,23\].

# Materials and methods

Two strains of *Eupenicillium brefeldianum*, (ATCC 10417 and 58665) were
obtained from the American Type Culture Collection. Both were grown in
shake flasks at 28°C for 7 days on four media: Czapek-Dox,
potato-dextrose, sorbitol-fumarate, and malt extract (MEM). MEM is
composed of glucose (2%), malt extract (0.2%), yeast extract (0.25%),
peptone (0.2%), KH~2~PO~4~(0.2%), MgSO×7H~2~O(0.2%), and CaCO~3~(0.4%),
pH 7.0.

![](media/image1.png){width="3.125in" height="2.6305555555555555in"}
Brefeldin-A was detected in the ethyl acetate extract of broth from ATCC
58665 grown on MEM. Studies were then undertaken to improve fermentation
conditions on MEM for the production of brefeldin-A. Stirring rate,
aeration, growth rate by centrifugal solids measurement, and glucose
utilization were monitored. Time-course monitoring for brefeldin-A titre
was done by hplc. Dow Corning type A (DCA)and PEG 1000 antifoams were
compared.

To prepare inoculum for the production phase, potato dextrose agar
slants were inoculated with 1.0 ml of glycerol vegetative stock which
had been stored at -70°C. The slants were incubated at 25°C for up to 96
hours or until confluent growth was obtained. These were washed with 5
ml of sterile MEM and the slant washings were used to inoculate the
first stage seed flask (100 ml culture medium/500 ml baffled Erlenmeyer
flask). The flasks were incubated at 25°C for 48 hours at 250 rpm on a
1\" stroke shaker. After xamining for purity under a microscope, the
second stage seed flask (1 L medium/4 L baffled Erlenmeyer flask) was
inoculated with a 5% volume. Again, the flasks were incubated for
48 hours at 25°C, 250 rpm.

After checking each flask for purity, the contents of several 4 L flasks
were pooled. The organism tended to grow in \'pellets\' and it was
determined that disruption of the pellets by homogenization in a
sterilized stainless steel Waring commercial blender, model CB-6, at
high speed for 2 minutes resulted in a more rapid growth rate following
transfer. Sufficient homogenate to give 5% v/v of inoculum was
transferred to a 100 liter fermentor containing 68 liters of MEM. Dow
Corning type A antifoam added at a level of 0.1% in the media in
fermentors of 100 liters and larger was effective in controlling the
frothing problem.

The incubation temperature was 25°C +/- 0.5, agitation rate 250 rpm
giving a tip speed of 327.0 ft/min. and air flow was 0.73 vvm. After
15 - 24 hours (mid-log phase), the contents of the 100 liter fermentor
were checked for culture purity, then transferred into a 1,100 liter
fermentor containing 680 liters of MEM. Fermentation was continued for
about 24 hours under the same conditions, at which point the entire
microbial culture was again checked for purity, then transferred into a
11,000 liter fermentor containing 6,800 liters of MEM. Operating
conditions were essentially unchanged for about 50 hours. The sequence
of events from initial vial through harvest, including a time line and
volume at each stage, is depicted in figure 2.

At harvest diatomaceous earth was added and the broth was filtered
through a pad of the same filter aid in a filter press. The clarified
broth was extracted once by vigorous stirring with a half volume of
ethyl acetate. Following concentration of the ethyl acetate extract to
one-tenth initial volume in a wiped-film evaporator and holding
overnight at room temperature without agitation, a fine, off-yellowish
precipitate formed that was removed by filtration through a coarse
filter paper. The remaining liquors were evaporated to dryness.

An analytical hplc method was developed which permitted near-real-time
monitoring of brefeldin-A production during the course of fermentation
(figure 3). Whole fermentation broth was extracted with an equal volume
of ethyl acetate, the organic solvent removed, dried under nitrogen,
resolubilized in methanol to a concentration of 1 mg/ml, filtered
through a 0.2 µm nylon filter (Rainin 38-109) and an appropriate volume
(usually 20 µl) injected onto a 4.6 x 150 mm, 3 µm C-18 phase bonded
silica column, (Rainin Microsorb \'Short One\'), fitted with a 3 µm C-18
4.6 x 15 mm guard column. All solvents were Burdick & Jackson hplc
grade. Isocratic elution with 57% methanol/43% water at 1 ml/min gave a
retention time of 5.8 minutes. A Waters 996 photodiode array detector
with the Waters Millennium 2010 software package was used to acquire and
analyze data. Standard curves were constructed at both 230 and 254 nm
with authentic brefeldin-A obtained from the DTP repository. The
standard was dissolved in methanol. Routine analysis was performed at
230 nm. Linearity was found between 0.3 and 250 mcg/ml at both
wavelengths. A quantification of brefeldin-A in crude extracts is
calculated by the chromatographic software from the values used to
construct the standard curve. This software additionally confirms
identity and provides a measure of purity of a compound at a preselected
retention time by comparison of the UV spectrum to the spectrum of a
standard.

An isocratic normal phase hplc method was developed using a 4.6 x 250
mm, 8 µm silica column (Rainin Dynamax 83‑101C) and guard column (Rainin
83-101G) eluted with 95% dichloromethane (DCM)/5% methanol at 1 ml/min.
Under these conditions brefeldin-A has a retention time of 11.2 minutes.
This method was used as an additional confirmation of purity of
crystalline material.

![](media/image2.png){width="4.0in" height="1.25in"}The presence of
brefeldin-A in broth extracts or column fractions can be qualitatively
determined by thin layer chromatography using glass-backed 0.25 mm
silica 60 plates (EM 5729-6), developed with either DCM:methanol (85:15,
Rf 0.50), or hexane:acetone (30:70, Rf 0.52). Visualization is
accomplished by spraying with 5% H~2~SO~4~ in 95% ethanol and heating
briefly with a hot air gun. Brefeldin-A turns a dark reddish color and
is detectable \<5 ng.

Large-scale flash chromatography was performed in a 4\" x 30\"
thick-walled borosilicate glass column (Kontes 584800-4000) using
Davison grade 22 silica chromatographic media, 60 x 200 mesh (Davison
Chemical Company, Baltimore, MD 21203). The silica was dry-packed, then
solvated by forcing pure DCM through at \<10 psi nitrogen pressure.
Liquors remaining after a first crop of crude crystals had been removed
were pooled and dried, giving 283 g of a dark brown semi-solid. The
entire pool was dissolved in DCM/methanol (1:1), mixed with twice its
weight of the same silica, rotary evaporated to dryness and vacuum dried
prior to overlayering onto 2,000 g of silica. Development was initiated
with DCM (10 L), and progressed stepwise through 1 (8 L), 2 (8 L), 3 (8
L), 4 (30 L), and 10 (2 L) percent methanol in DCM, with flow controlled
by the application of nitrogen pressure. The DCA was eluted by DCM,
followed by brefeldin-A enriched material when the solvent composition
reached 4% methanol in DCM. Following evaporation to dryness and
resolubilization in warm methanol, setting in a refrigerator overnight
yielded crystalline brefeldin-A.

Brefeldin-A is remarkably stable to acid and base but following
treatment of extracted broth or equipment used in production in aqueous
0.25% sodium hypochlorite solution for 1 hour at ambient temperature,
none of the title compound could be detected by hplc analysis.

Results

A crystalline substance isolated from the ethyl acetate extract of ATCC
58665 grown on MEM in a small shake flask was shown to be
7-(S)-brefeldin-A by thin layer, hplc, NMR, MS and optical rotation (OR)
by comparison to an authentic standard. The literature value given for
the OR of 7-(S)-brefeldin-A is +82° in ethyl acetate compared with our
measurement of +85° in methanol. Strain 10417 did not produce the
desired compound, and no effort was made to determine if brefeldin-like
compounds \[7,28,33\] or other bioactive substances \[17,19\] were
present in the extract from either organism.

Having determined that the desired compound was being produced by ATCC
58665, trial fermentations on MEM in 10 liter stir jars were performed
until consistent production of brefeldin-A at a titre greater than 100
mg/L in \<90 hours was attained. This was considered satisfactory for
further scale-up. ATCC 58665 grew luxuriantly, with a yellow pigment
sometimes observed \[15\]. However, the appearance of the yellow pigment
did not correlate with brefeldin-A production.

Experience was gained through 9 progressively larger-scale fermentations
(multiple 10, 100 and 1,100 liter fermentors) which lead to successful
completion of four fermentations of 6,800 liters each. Data typical of a
fermentation at that scale is presented here.

The titre of brefeldin-A was observed to begin climbing sharply
approximately 20 hours following inoculation of the 11,000 liter tank,
and to continue rising to a peak after about 50 hours of fermentation
(figure 2). During developmental runs, a slow decrease in brefeldin-A
titre was observed to occur in the 12 hours following the peak titre if
fermentation was allowed to continue. This was coincident with a
decrease in pH and in centrifugal solids (packed cell volume), both of
which could also be used as alternative indicators for harvest. A
consistent decrease in pH was observed during the course of
fermentation. A pH below 5.0 was deleterious to the optimum production
of brefeldin-A. The presence of 0.4% calcium carbonate in the production
medium maintained the pH above 5.0 making upward adjustment by addition
of base unnecessary.

Analysis by hplc showed that 92% of the desired compound was removed
from the broth by a single extraction with ethyl acetate, and that
typically \>60% of the mass of this extract was brefeldin-A. The
off-yellow precipitate obtained from the ethyl acetate extract following
concentration, 1,123 g, was recrystallized twice from methanol to yield
1,026 g of large, white crystals of brefeldin-A. Analysis by both normal
and reverse phase hplc showed these crystals to be \>99% of the title
compound.

Column chromatographic purification of brefeldin-A from the solids
remaining in the pool of liquors following crystallization (283 g)
yielded an additional 120 g of the desired compound. Isolated yield in
this instance was 1,146 g of brefeldin-A from 6,800 liters of broth, or
169 mg/L.

Discussion

A practical method for the production of kilogram quantities of the
anticancer antibiotic brefeldin-A by liquid fermentation has been
reported. The isolated yield of brefeldin-A, 169 mg/L, was less than
that reported (278 mg/L) for stationary culture on a potato dextrose
medium \[25\], but scale-up of that method to provide a large quantity
of the desired compound, though possible, would be considerably more
difficult than the method reported here. Liquid fermentation at the 50
liter scale on a medium similar to MEM resulted in a yield of \<15 mg/L
\[10\].

The determination of optimum fermentation conditions for the production
of brefeldin-A was greatly facilitated by the development of a rapid
hplc assay which allowed for quantitation of the desired compound from 3
ml of whole fermentation broth within 2 hours of the time a sample was
taken. As this project progressed from shake flasks to fermentors of
increasingly greater volume, the rapid hplc assay provided important
information indicating that drug titre in the broth climbed faster and
reached a peak earlier in larger vessels than it had in smaller
fermentors. The ease and speed at which a brefeldin-A titre profile
could be constructed through use of the hplc assay permitted harvest to
occur at very nearly the peak of production, thus, yield of the desired
product could be maximized.

Extraction of brefeldin-A with a half-volume of ethyl acetate from the
clarified broth was highly effective, giving, after concentration, a
first crop of crude brefeldin-A solids equal to 89% of the isolated
yield. Considering the time, labor and materials expended in
chromatography, a careful evaluation of the economics of purifying a
rather small additional quantity of product from liquors after
crystallization should be made in any future large-scale production of
brefeldin-A. If testing of this compound by the National Cancer
Institute demonstrates its usefulness as an adjunct in cancer
chemotherapy, further scale-up of fermentative production of brefeldin-A
should be possible with little modification of the techniques reported
here.

Acknowledgements

The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organization imply
endorsement by the U.S. Government.

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Legend to the figures

Fig. 1 Structure of 7-(S)-brefeldin-A

Fig. 2 Scheme for scale-up production of brefeldin-A to an 11,000 liter
fermentor providing a time line and volume at each stage.

Brefeldin-A, (synonyms: ascotoxin, cyanein, decumbin, lunatin) a
13-carbon macrolide lactone antibiotic first isolated by