Note: Descriptions are shown in the official language in which they were submitted.
~04~;0SZ
PROCESS FOR THE PURIFICATION OF FR-1923 SUBSTANCE
The present invention relates to a new process for
purification of FR-1923 substance. ~lore particularly, it
relates to a new process for purification of FR-1923 sub-
stanee, using a macroporous non-ionic adsorption resin.
FR-1923 substance is a known antibiotie isolated from
the fermentation broth of a strain of genus Stre~tomvce~,
as described, for example, in German Offenlegungsschrift
2242699 corresponding to Canadian Patent Application
Ser.~o. 150,731 filed August 30,1972 Hatsuo Aoki et al.
In said prior literature, there are disclosed processes
using aetivated ehareoal, anion exehange resin and DEAE-
Sephadex (trade mark, made by Pharmacia Co.) for isolation
and purification of FR-1923 substance from a fermentation
broth.
However, the prior proeesses have not led to satisfae-
tory results in an industrial manufacture of FR-1923 sub-
stanee beeause they require relatively many steps for the
isolation and purifieation of FR-1923 substanee, so that
the overall recovery of the antibiotic is relatively poor.
As the results of the extensive studying, the present
inventors have now newly found a new process for purification
of FR-1923 substance as a step in the recovery thereof, by
means of which FR-1923 substance can be obtained in higher
yield than by the prior processes. Further, this new process
is simpler and more convenient in practical operation and
-- 1 --
1046()52
more economical on an industrial scale because the spent resin
can be regenerated to be used again.
Accordingly, it is an object of the present invention
to provide a new process for purification of FR-1923 substance
usinq a macroporous non-ionic adsorption resin.
Another object of the present invention is to provide
a new process for purification of FR-1923 substance, by means of
which FR-1923 substance is obtained in higher yield than by the
prior processes.
A further object of the present invention is to provide
a simpler, more convenient and more economical process for puri-
fication of FR-1923 substance.
According to the invention there is provided a process
for the purification of FR-1923 substance which comprises:
contacting an aqueous solution containing FR-1923 substance,
together with impurities, with a macroporous nonrionic adsorption
resin, adsorbing one of said FR-1923 Aubstance and said impurities
in ~aid solution on said re~in, and recovering said FR-1923 sub-
stance.
In one aRpect of the invention the aqueous solution is
contacted with the macroporous non-ionic adsorption resin to
adsorb FR-1923 substance on the resin, the adsorbed FR-1923
substance i~ eluted from the re~in, and the FR-1923 substance is
recovered from the eluate.
In another aspect of the invention the aqueous
solution is contacted with a macroporous non-ionic adsorption
resin saturated with FR-1923 substance, impurities are adsorbed
on the saturated resin, and FR-1923 substance is recovered from
the thus contacted solution.
As described above, the present invention provides a
new process for purification of FR-1923 substance, using a
macroporous non-ionic adsorption resin, which comprises alter-
k
~ Jl ~ ~ 2 -
r, ~,
104605Z
native methods in u~ing a macroporous non-ionic adsorption resin:
That is, one of them is a method that an aqueous solution
containing FR-1923 substance, together with impurities, is
contacted with a macroporous non-ionic adsorption resin,
whereafter the adsorbed FR-1923 substance is eluted with,
for example, a hydrophilic solvent system (hereinafter con-
veniently referred to method A), The other is a method that an
aqueous solution containing FR-1923 substance, together
with impuritie~, is contacted with a macroporous non-ionic
adsorption resin saturated by FR-1923 substance to adsorb
the impurities, and FR-1923 substance is recovered from the
B- 2a -
104605Z
passed solution (hereinafter conveniently referred to method B).
Alternative methods are explained in more detail hereinafter.
M~thod A
According to method A of the present invention, there
is provided a process for purification of FR-1923 substance,
wherein an aqueous solution containing FR-1923 substance,
together with impurities, is contacted with a macroporous
non-ionic adsorption resin, whereafter the adsorbed FR-1923
substance is eluted with, for example, a hydrophilic solvent
system.
.
The macroporous non-ionic adsorption resins used
according to the present invention may include a cross-linked
. resin with an aromatic basic structure. The preferred
resins include styrene polymers cross-linked with divinyl
benzene. Examples of such resins include Amberlite XAD-l,
XAD-2, XAD-4, XAD-7 and XAD-8 (trade mark, maker; Rohm &
Haas Co.), Diaion HP 10, HP 20, HP 30, HP 40 and HP 50
(trade mark, maker; Mitsubishi Kasei Co., Ltd.) and Hitachi-
gel No. 3010 (trade mark, maker; Nissei Sangyo Co., Ltd.).
The process for purification of FR-1923 substance,
using the macroporous non-ionic adsorption resin, in accord-
ance with the method A of the present invention is applicable
to any aqueous solution containing FR-1923 substance,
together with impurities. Examples of such solutions include
a filtrate of culture broth per se prepared by cultivating
FR-1923 substance - producing microorganism in a nutrient
medium, and a pre-extracted culture solution as well as an
aqueous solution containing crude solid material containing
10460S2
F'R-1923 substance (e.g. crude powder or crude crystals
containing FR-1923 substance) which first is isolated and
purified to some degree from a filtrate of the culture
broth by means of conventional manners, such as an optional
treatment of the filtrate with an appropriate solvent for
extraction and/or by adsorption on activated charcoal.
When using a culture filtrate, it is advantageous to
use an optionally pre-extracted culture solution, this
optionally pre-extracted culture solution being brought
into contact with a macroporous non-ionic adsorption resin
in the usual manner, preferably with use of a column
containing a bed of resin.
Treatment of the aqueous solution containing FR-1923
substance in admixture with impurities with the non-ionic
adsorption resin is advantageously carried out at pH value
of from 2-7 and preferably of from 4 to 6. The desired
acidic pE~ values can be achieved by using any desired acid,
for example, an organic acid, such as oxalic acid, or
preferably a mineral acid, such as hydrochloric, phosphoric
or sulphuric acid.
Further, treatment of the aqueous solution containing
FR-1923 substance in admixture with impurities with the
non-ionic adsorption resin is advantageously carried out in
case of the impurities being inorganic salt, such as sodium
chloride, sodium biphosphate or disodium orthophosphate.
In this method, the aqueous solution containing FR-1923
-- 4 --
1046Qsz
substance in admixture with impurities is brought into
contact with the macroporous non-ionic adsorption resin to
adsorb FR-1923 substance by a conventional manner, for
example batch operation or column operation, whereafter
adsorbed FR-1923 substance is eluted from the resin with,
for example, a hydrophilic solvent system.
The hydrophilic solvent systems used for eluting FR-1923
substance from the resin, include, for example, a lower
dialkyl ketone (e.g. acetone and methyl ethyl ketone); and
a lower alkanol (e.g. methanol, ethanol, n-propanol, iso-
propanol, n-butanol and isobutanol). There may also be used
mixtures of the above lower dialkylketone and lower alkanol.
Further, mixtures of watér or of a lower alkyl ester of a
lower alkanoic acid, for example methyl acetate, ethyl
acetate and butyl acetate, and the above lower alkanol or
lower dialkyl ketone may also be used. Furthermore, a
large volume of water and an alkaline aqueous solution
Ipreferably pH 8 - 13) may be used.
The eluate thus obtained is treated by conventional
means such as concentration, pH adjustment, lyophilization
and recrystallization to give crystalline FR-1923 substance
which frequently is sufficiently pure for use as a medicine.
Method B
According to method B of the present invention, there
is provided a process for purification of FR-1923 substance
wherein an aqueous solution containing FR-1923 substance,
together with impurities, is contacted with a macroporous
1046052
non-ionic adsorption resin saturated in advance by FR-1923
substance to adsorb impurities, and FR-1923 substance is
recovered from the passed solution.
Thi~ method is based on the fact that adsorption
capacity of a macroporous non-ionic adsorption resin to
FR-1923 substance is smaller than that to specific impurities
such as coloring materials stemming from fermentation broth
and isomer of FR-1923 substance. By using the
difference of capacity of macroporous non-ionic adsorption
reqin between FR-1923 substance and the specific impurities,
the specific impurities are selectively adsorbed to the
resin saturated by FR-1923 substance from an aqueous
solution containing FR-1923 substance, together with im-
purities, and FR-1923 substance is recovered from the passed
solution.
Ad~orption capacity of a macroporous non-ionic
adRorption resin to ~R-1923 substance varies with condition
of aqueous solution containing FR-1923 substance, together
with impurities, and with the kind of the macroporous non-
ionic adsorption reqin. Exampleq of adsorption capacity of
a macroporous non-ionic adQorption reqin to FR-1923 sub-
stance are as follows. The following data is given by
contacting 0.2M phosphate buffer solution ~H4) containing
FR-1923 substance with the resin.
Examples of adsorPtion caPacitY to FR-1923 substance
Ca . lOg/l~ of Diaion HP 20*
Ca. 18g~1~ of Amberlite XAD-4*
*trademark
t . - 6 -
iO460S2
The macroporous non-ionic adsorption resin saturated
by FR-1923 substance is prepared by contacting an aqueous
solution containing FR-1923 substance with a macroporous
non-ionic adsorption resin, wherein said solution is
preferably neutral or slightly alkaline.
As examples of a macroporous non-ionic adsorption resin,
the same examples as mentioned in the method A are exemplified.
The process for purification of FR-1923 substance
using the macroporous non-ionic adsorption
resin, in accordance with the method B of the present
invention is applicable to any aqueous solution containing
FR-1923 substance, together with impurities, as well as that
of method A mentioned above, as examples of which the same
examples as mentioned in the method A are exemplified.
Especially, when coloring materials or isomer of
FR-1923 substance are contained as impurities in the aqueous
solution,thls method B gives better results.
When an aqueous solution containing FR-1923 substance,
together with impurities, is contacted with the macroporous
non-ionic adsorption resin saturated by FR-1923 substance,
FR-1923 substance can be adsorbed no more to it and, on the
other hand, the impurities are selectively adsorbed to it.
Volume of the macroporous non-ionic adsorption resin
to be used in this method varies with a content of FR-1923
substance in an aqueous solution containing FR-1923 substance,
together with impurities, and with the kind of the resin to
be used, and it, generally, may be lQ per 100 - 500 g of
-- 7 --
1046052
FR-1923 substance in the aqueous solution.
FR-1923 substance is recovered from the passed through
solution, which is obtained by contacting an aqueous solution
containing FR-1923 substance, together with impurities, with
macroporous non-ionic adsorption resin saturated by FR-1923
substance. The recovery can be conducted by conventional
manners such as pH adjustment, treatment with activated
charcoal, concentration, lyophilization and recrystallization
to give crystalline FR-1923 substance which frequently is
sufficiently pure for use as a medicine.
Further, an aqueous solution containing FR-1923
substance, together with impurities, is contacted with a
macroporous non-ionic adsorption resin saturated by FR-1923
substance to adsorb impurities, whereafter the column is
washed with a small volume of water to obtain FR-1923 substance
staying of the space among the resin in the column.
The spent resin can be regenerated by conventional
manners, e.g. by washing the said resin with a relatively
strong alkaline aqueous or alkaline aqueous-alcoholic solution.
The method B is superior to the method A and is more
convenientin practiceand more economical on an industrial
scale, in the following points. That is, the method B does
not require a process for elution of FR-1923 substance from
the resin, and volume of the resin used for purification in
method B is less than 10% in comparison with that in method
A, though recovery and purity of FR-1923 substance are almost
1046~SZ
same degree in both methods.
The following testsare given for the purpose of illustra-
ting a comparison between method A and B as a process for
~urification of FR-1923 substance.
The comparison-tests are carried out by using the same
crude crystals of FR-1923 substance and the same resin in
` the same volumein both methods, but volume of the crude
crystals of FR-1923 substance in the method A is decided
within the capacity of the resin to FR-1923 substance, and
volume of the crude crystals of FR-1923 substance used in
the method B is about 20 times that in method A.
Test
A lI] Adsorption capacity of Diaion HP 20 to FR-1923 substance:
1.4 g of sodium salt of FR-1923 (purity : 95%) substance
were dissolved in 1 liter of 0.2 M phosphate buffer solution
(pH 4.0). The solution was adjusted to pH 4.0 with 5N
hydrochloric acid with stirring. The solution (concentration
of FR-1923 substance : 1310 r/ml) was passed through a column
packed with 100 ml of Diaion HP 20 (internal diameter of the
column : 30 mm, packing height of the resin : 160 mm) at a
rate of SV=l ("SV" means abbreviation of "Space Velocity").
The passed solution were collected lOOml by lOOml. Content
of FR-1923 substance in the passed solution was determined
by bioassay using Pseudomonas aeruginosa. Adsorption capacity
of Diaion HP 20 to FR-1923 substance was calculated at the
time when concentration of FR-1923 substance in the passed
solution reaches the same concentration as that of the
~ ~r~o~ark
g
1046QS2
starting solution (1310 r/mQ~ to give 10.4 g / lite~.
[II] Purification of FR-1923 substance by the method A:
9.5 g of crude crystals of FR-1923 substance (FR-1923
substance content : 7.9 g) were suspended in 2.5 liters of
water. The suspension was adjusted to pH 7.5 with 6N sodium
hydroxide aqueous solution to dissolve in. Further, the
solution was adjusted to pH 6.0 with 6N hydrochloric acid,
and to the solution there were added 150 g of sodium
chloride and water to give 3 liters of the solution. The
solution was passed through a column packed with 1 liter of
Diaion HP 20 (internal diameter of the column: 77 mm, packing
height of the resin: 230 mm) at a rate of SV=l. The adsorbed
FR-1923 substance was eluted with 30% aqueous methanol after
the column was washed with water. Active fraction (1950 ml)
were collected and concentrated under reduced pressure. The
concentrate was adjusted to pH 2.2 with stirring and allowed
to stand overnight in the refrigerator to give crystals.
The crystals were collected by filtration and dried. The
results are shown in the following table.
lIII] Purification of FR-1923 substance by the method B:
Two hundred gram of crude crystals of FR-1923 substance
(FR-1923 substance content : 168 g) being the same crude
crystals as that used in Test [II], were suspended in 2
liters of water. The suspension was adjusted to pH 7.2 to
A~ dissolve in. The solution was passed through a column packed
;~ with 1 liter of Diaion HP 20 saturated by FR-1923 substance
(internal diameter of the column : 77 mm, packing height of
the resin : 230 mm) at a rate of SV=l. Four liters of water
~rao~ n a~k
-- :LO --
.. .. . .
10460S;~
were passed through the column, 4500 ml of active fractions
were collected from the passed solution, and to the solution
obtained there was added water to give 16 liters of the
solution. The solution was àdjusted to pH 2.5 with stirring
and then allowed to stand overnight in the refrigerator to
give crystals. The crystals were collected by filtration
and dried. The results are shown in the following table.
The method A The method B
Capacity of the resin
for treating crude 9.5 g / lQ of 187 g / lQ of
crystals of FR-1923 resin resin
substance
Recovery of crystals 1 7.2 g 160 g
Purity of crystals 92.7 % 98.4 %
Purity-calcd,recovery 84.5 % ¦ 93.6 %
of FR-1923 substance
The above results show that volume of FR-lg23 substance
treated by method B is about 20 times that by method A,
though purity and recovery of FR-1923 substance are almost
the same degree in both methods, respectively.
The following examples are given for the purpose of
illustrating the present invention.
1046052
Example 1
To a culture broth prepared by culturing Nocardia
~niformis var. tsuyamanensis ATCC 21806 at 30C for 120 hours
in 150 liters of a nutrient medium containing by weight 3%
glycerol, 2% cottonseed meal, 2% dried yeast, 2.18% potassium
dihydrogen phosphate, 1.43% disodium hydrogen phosphate
dodecahydrate, 0.5% magnesium chloride heptahydrate, there
was added 6% of Radiolite (trade mark, a filter aid material
sold by Showa Kagaku Kogyo Co., Ltd.) and then the mixture
was filtered. Three liters of the filtrate obtained
(FR-1923 substance content: 3030 mg) were passed through a
A column packed with a macroporous non-ionic adsorption resin,
Diaion HP 20 (internal diameter of the column : 8 cm, packing
. height of the resin : 26 cm) at a rate of SV=l. The column
was washed with water, whereafter FR-1923 substance was
eluted with 20% of aqueous methanol solution. Three liters
of eluate obtained were concentrated under reduced pressure
and then adjusted to pH 2.5. The solution was allowed to
stand in refrigerator to give 2490 mg of colorless crystals
of FR-1923 substance.
Example 2
A culture broth which was prepared by substantially the
same manner as described in Example 1 was adjusted to pH 4.0
with diluted sulfuric acid, whereafter the culture broth was
filtered. To the filtrate obtained was added 6% of Radiolite,
whereafter the mixture was filtered. Nine hundred ml of the
filtrate obtained (FR-1923 substance content : 774 mg) were
passed through a column packed with a macroporous non-ionic
adsorption resin, Amberlite XAD-4(internal diameter of the
t ~a O~ ark
- 12 -
1046C)52
column: 3.6 cm, packing height of the resin~30 cm) at a rate of
SV=l. After the column was washed with water, FR-1923 sub-
stance was eluted with 20~ aqueous acetone. Six hundred ml
of eluate obtained were concentrated under reduced pressure,
adjusted to pH 2.0 with diluted hydrochloric acid, and then
allowed to stand in a refrigerator to give 365 mg of
colorless crystals of FR-1923 substance.
Example 3
Three hundred ml of a culture filtrate (pH`4.0),
obtained by substantially the same manner as described in
Example 1, were passed through a column of a macroporous non-
A~ ionic adsorption resin, Diaion HP 20 (internal diameter of
the column : 30 mm, packing height of the resin : 160 mm)
at a rate of SV=l. The column was washed with water,
whereafter FR-1923 substance was eluted with aqueous sodium
hydroxide solution (pH 12.0). Six hundred ml of active
fraction obtained were collected and concentrated to 15 ml
under reduced pressure. The concentrate was adjusted to
pH 2.5 with 6N hydrochloric acid while stirring, whereafter
the solution was allowed to stand for 24 hours in the
refrigerator to give 360 mg of colorless crystals of FR-1923
substance.
Example 4
Twenty g of crude powder (FR-1923 substance content :
10.2 g) of FR-1923 substance were suspended in 6.2 Q of
water. The suspension was adjusted to pH 8.1 with 6N aqueous
sodium hydroxide solution, whereafter 50 g of sodium chloride
were added to the solution; The solution was passed through
~ ~ r6(a~ h?ark
- 13 -
~046~52
a column of a macroporousnon-ionic adsorption resin, Diaion
HP 20 (internal diameter of the column : 87 mm, packing
height of the resin : 520 mm) at a rate of SV=l. The column
was washed with water, whereafter FR-1923 substance was
eluted with 30% aqueous methanol solution. Active fractions
(4.48 liters) obtained were collected and then concentrated
to the volume of 400 ml under reduced pressure. The
concentrate was adjusted to pH 2. 5 with 6N hydrochloric acid
and then allowed to stand overnight in refrigerator to give
10.0 g of colorless crystals of FR-1923 substance.
Example 5
Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of
water, whereafter the suspension was adjusted to p~I 7.0 with
6N sodium hydroxide aqueous solution to dissolve in. The
solution was passed through a column packed with a macroporous
non-ionic adsorption resin, Amberlite XAD-8 (internal
diameter of the column : 30 mm, packing helght of the resin :
160 mm) saturated by FR-1923 substance at a rate of SV=l,
whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed
solution, and then the solution was concentrated under reduced
pressure to a volume of 200 ml. The concentrate obtained
was adjusted to pH 2. 5 with 6N hydrochloric acid while
stirring, and then allowed to stand in a refrigerator for
24 hours to give 10.13 g of colorless crystals of FR-1923
substance.
~ ~ æhn~ r ~
- 14 -
. .
1046052
Example 6
Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of water
and adjusted to pH 7.0 with 6N sodium hydroxide aqueous
solution to dissolve in. The solution was passed through a
column packed with a macroporous non-ionic adsorption resin,
Amberlite XAD-4 (internal diameter of the column : 30 mm,
packing height of the resin : 160 mm) saturated by FR-1923
substance at a rate of SV=l, whereafter 540 ml of water were
passed through the column. 900 ml of active fractions were
collected from the passed solution, and then the solution
was concentrated under reduced pressure to a volume of 350
ml. The concentrate obtained was adjusted to pH 2. 5 with
6N hydrochloric acid while stirring and then allowed to
stand in a refrigerator for 24 hours to give 9. 41 g of
colorless crystals of FR-1923 substance.
Example 7
Nineteen gram of crude power (FR-1923 substance content:
10. 7 g) of FR-1923 substance were suspended in 380 ml of
water and then the suspension was adjusted to pH 7.0 with
6N sodium hydroxide aqueous solution to dissolve in. ~he
solution was passed through a column packed with macroporous
non-ionic adsorption resin, Amberlite XAD-7 (internal
diameter of the column: 30 mm, packing height of the resin:
160 mm) saturated by FR-1923 substance at a rate of SV=l,
whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed
solution, whereafter the solution obtained was adjusted to
pH 2. 5 with 6N hydrochloric acid while stirring and a~lowed
~ ~a6~QrnQrk
1046052
to stand in a refrigerator to give 10.28 g of colorless
crystals of FR-1923 substance.
Example 8
19.75 g of crude powder of FR-1923 substance (FR-1923
substance content: 17.16 g) were suspended in 395 ml of
water and the suspension was adjusted to pH 7.5 with 5N
sodium hydroxide aqueous solution to dissolve in. The
solution was passed through a column packed with a macroporous
1 A ~ non-ionic adsorption resin Diaion HP 20 (internal diameter
of the column : 30 mm, packing height of the resin : 160 mm)
saturated by FR-1923 substance at a rate of SV=l, whereafter
400 ml of water were passed through the column. 600 ml of
active fractions were collected from the passed solution~
whereafter the solution obtained was adjusted to pH 2.5 with
6N hydrochloric acid while stirring and allowed to stand in
a refrigerator to give 16.8 g of colorless crystals of
FR-1923 substance.
Example 9
Fifteen liters of a culture filtrate (pH 4.0)
(FR-1923 substance content: 14.25 g), obtained by substantially
the same manner as described in Example l,were passed
through a column packed with a macroporous non-ionic
adsorption resin, Diaion HP20 (internal diameter of the
coulum: 110 mm, packing height of the resin: 550 mm) at
a rate of SV=l. The column was washed with 5 liters of
water, whereafter FR-1923 substance was eluted with 30~
aqueous methanol solution. 7,940 ml of active fractions
obtained were collected and then adjusted to pH 3.3 with
* ~ 1ar ~
- 16 -
10460S2
6N hydrochloric acid. The solution was passed through
a column of alumina (acidic, internal diameter of the
column: 46 mm, packing height of alumina: 300 mm).
The column was washed with 500 ml of water, whereafter
FR-1923 substance was eluted with 0. 3 M pottassium
biphosphate aqueous solution. 1800 ml of active fractions
were collected and then concentrated under reduced
pressure to the volume of 800 ml. The concentrate was
adjusted to pH 2.2 with 6N hydrochloric acid while
stirring, whereafter the solution was allowed to stand
overnight in a refrigerator to give 11.94 g of colorless
crystals of FR-1923 substance.
Example 10
Thirty liters of a culture filtrate (pH 4.0) (FR-1923
substance content: 32.4 g), obtained by substantially
the same manner as described in Example l,were passed
through a column packed with a macroporous non-ionic
adsorption resin, Diaion HP20 (internal diameter of the
column: 140 mm, packing height of the resin: 600 mm)
at a rate of SV=l. The column was washed with 10 liters
of water, whereafter FR-1923 substance was eluted with
0.01 N sodium hydroxide aqueous solution. 30.1 liters of
active fractions were collected and then adjusted to pH 3.3
with 6 N hydrochloric acid. The solution was passed
through a column of alumina (acidic, internal diameter
of the column: 77 mm, packing height of alumina: 440 mm).
The column was washed with 2 liters of water, whereafter
FR-1923 substance was eluted with 0.3M potassium biphosphate
aqueous solution. 12. 3 liters of active fractions were collected
k -17 -
1046~5Z
and then concentrated under reduced pressure to the
volume of 1,600 ml. The concentrate was adjusted to
pH 2.2 with 6N hydrochloric acid while stirring, whereafter
the solution was allowed to stand overnight in a
refrigerator to. give 25.95 g of colorless crystals of
FR-1923 substance.
.
- 18 -
