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Patented Mar. 16, 1948 ,
2,437,918
UNITED STATES PATENT OFFICE,
2,437,918
METHOD OF PRODUCING PENICILLIN
Robert B. McCormack, Milltown, N. 1., asslgnor
to E. R. Squibb 8: Sons, New York, N. Y., a
corporation of New York
No Drawing. Application January 21, 1944,
1
Serial No. 519,207
4 Claims. (Cl. 195-36)
This invention relates to the production of
penicillin, an antimicrobial agent which has beenv
found exceptionally effective as a therapeutic.
Penicillin has heretofore been produced by
growing a penicillin-forming mold, especially
PeniciZlium notatum, on the surface of a liquid
2
place of the corn steep liquor (and especially if
dextrose or other pure substance is used as the
source of carbohydrate) an efficient liquid nutri
ent medium is obtained which is wholly syn
thetic.
The term “sul?te-type compound,” as employed
herein, has reference to the genus composed of
compounds embodying a sul?te radical and com
nutrient medium. More recently, the mold has
been grown in submerged culture, 1. e., by in
cubating the mold while it is submerged in a liq
pounds capable of decomposition into compounds
uid nutrient medium and the latter is aerated, 10 embodying a sul?te radical. This genus is rep
by agitation or other suitable means; which
resented by the sul?tes, bisul?tes, metabisul?tes,
method is obviously better adapted for the large
hyposul?tes (i, e., salts of hyposulfurous acid),
scale commercial production of penicillin,
and thiosulfates; and it includes, inter alia, so
Liquid nutrient media of various compositions
dium sul?te, potassium sul?te, ammonium sul
have been employed heretofore in the production 15 ?te, magnesium sul?te, calcium bisul?te, sodium‘
of penicillin by mold growth, the media generally
comprising nutrient salts and carbohydrate as
similable by the mold. In the ‘.production of
penicillin by submerged culture, it has been
metabisul?te, sodium hyposul?te, sodium thio
sulfate, and sodium bisul?te. The sul?te
(and/or bisul?te) may be formed in situ by add~
ing sulfurous acid or by passing S02 into the me
found that the inclusion of a complex organic 20 dium; and industrial by-products rich in inor
mixture of nitrogenous substances—especlally
corn steep liquor-increases the efficiency of
penicillin production by causing a more rapid
growth of the mold, higher titers, and attain
ganic, water-soluble sul?te-type compounds,
such as sul?te waste liquor, may be employed
(not, however, conferring the advantages of a
wholly synthetic medium) .
ment of maximum penicillin production in a 25
Though certain batches of corn steep liquor
shorter time. The use of such complex mixture,
and other complex organic mixtures of nitrog
however, is attended by a number of disadvan
enous substances may contain a minute propor
tages arising from the lack of uniformity in the
tion of an inorganic, water-soluble sul?te-type
composition of mixtures from different batches
compound, their use for the purpose of including
and sources; thus, the lack of uniformity intro
an inorganic, water-soluble sul?te~type com
30
duces obviously undesirable variations in the
pound in the medium would require pretesting of
penicillin content of the culture ?ltrate, and the
each batch and is therefore out of the question.
_ ,heterogenicity and complexity of the components
"of, such mixtures give rise to difficulties in the
extraction and concentration of the penicillin,
“Synthetic” liquid nutrient media (i. e., liquid nu—
trient media prepared only with ingredients of
Desirably, the amount of inorganic, water-sol
uble sul?te-type compound included in the me
dium is that theoretically capable of yielding a
proportion of sul?te radical to medium of about
0.02% to about 0.65%, preferably about 0.033.
known, controllable, and reproducible composi
0.4%, and for optimum results (especially when
tion) may be employed, but production there
2% sugar is included) about 0.065-0.2%.
with has been ine?lcient.
Using the improved media and incubation
It is the object of this invention to provide im 40 methods of this invention, highly-potent, non
proved methods of and media for producing pen
toxic, sodium-penicillin ‘preparations, for exam
icillin by mold growth, especially by submerged
ple, may be obtained by the following general
procedure:
culture; and it is an additional object of this
invention to provide methods of and media. for
A liquid nutrient medium in which has been
e?iciently producing penicillin by mold growth 45 included an inorganic, water-soluble sul?te-type
utilizing synthetic liquid nutrient media.
compound is sterilized (in the usual manner),
It has been found that the inclusion of an in
inoculated with (spores of) a selected strain of
organic, water-soluble sul?te-type compound in
the mold Penicillium notatum, and incubated un
the liquid nutrient medium greatly increases the
til substantially maximum penicillin production
ei?ciency of penicillin production, and that the 50 has been attained; and the culture medium is ?l
inclusion of such compound obviates the use of
corn steep liquor (or other complex organic mix
ture of nitrogenous substances) with its attend
ant ‘disadvantages, Thus, if an inorganic, water
tered, the ?ltrate is acidi?ed and extracted with
amyl acetate in the cold, the amyl acetate solu
tion is extracted with a pH 6.5 phosphate-buffer
, solution, the buffer solution is- cooled, acidi?ed,
soluble sul?te-type compound is included in 55 and extracted with chloroform, the chloroform
2,437,918
3
soultion is extracted with a pH 6.0 phosphate
buffer solution, and the cooled bu?’er solution is
acidi?ed and then extracted with ether, and the
ether solution is extracted with sodium bicarbon
ate solution. The resulting solution of sodium
penicillin is frozen and subjected toa high vac
uum to sublime out the water.
i
The following examples are ‘illustrative of the
invention (the potency unit referred to being
the Florey, or Oxford, unit used in the United
States of America for standardizing'therapeutic
penicillin preparations) :
Example "1
I
‘
'
., _
l
'
'
‘
4
culture ?ltrate being 8.8. (An improvement in
rate of penicillin production may be obtained by
replacing the sodium nitrate in the formula with
about 6-12 g. potassium nitrate; thus, with the
same formula but including 6 g. potassium ni
..tratein place of the sodium'nitrate, and under
.the same conditions, a maximum potency of 36
units/ml. is developed in 4 days, the pH of the
culture ?ltrate being 8.2.)
Example 6
Ten gallons of a tap-water medium made up
‘ with the following percentages of ingredients:
. -
Na'zSOs _
150 cc. portions of a medium of the followin
formula:
‘g
NaNO; _____________________________ _-g__
KHEO4
‘
2"
MgSOaK ' 'IHzO _______________________ __g__
CaCOa
-
g
KHZPOL
.
'6
_ 0.15
-__
0.15
MgSOr'lHzO __________________________ __ 0.05
NaNOa _________________________________ __ 0.6
1.5
CaCO: ___
0.5
Brown sugar __________________________ _.. 2.0
5
Brown sugar _______________________ __g-_' 10
NazSOa ____________________________ _-g__
1
Tap water _______________________ __liter__
1
are distributed in 500 cc. ?asks and sterilized, an
_
_.. 0.5
20 is‘placed in a tank and sterilized,‘ an inoculum of
- 'Pen‘icillium notatum is added, and the medium is
incubatedat 25-26‘ C. while‘maintained‘ under
an air pressure ‘of about 7 lbs_., with air passing
into the medium through a sparger. The tank is
harvested after 3 days’ incubation, the culture
?ltrate obtained having a potency of 12 units/ml.
inoculum of Peniclllium notatum is added to
each ?ask, and the medium is incubated at 75°
F, while agitating continuously by means of a
Example 7
shaking machine. A maximum potency of 12
Employing
a
tap-water
medium made up with
units/ml. is developed in 4 days, the pH of the 30
the
following
percentages
of ingredients:
culture ?ltrate being 8.0. (With the sodium sul
?te omitted from this formula, and under the
NazSO: _______________________________ .. ..
0.2
same conditions, a maximum potency of 4.6
KH2PO4 ______________________________ __
0.2
units/ml. is developed in 4 days, the pH of the
MgSOr'lHzO ______________________ _-'____ 0.1
culture ?ltrate being 7.8.)
_ 0.6
35 NaNOa
CaCOs
_.. 0.5
Example 2
Brown sugar
-..___
2.0
With the sodium sul?te in the formula of Ex
in place of the medium in Example 6, and under
ample 1 increased to 2 g., and under the same
the same conditions, a maximum potency of 12
conditions, the same maximum potency is de
veloped in the same time, but the pH of the cul 40 units/ml. is developed in 5 days, the pH of the
culture ?ltrate being 8.3. The sodium-penicillin
ture ?ltrate is 9+.
obtained from this ?ltrate in the manner detailed
Example 3
hereinbefore is a light-yellow, fluffy, non-hygro
Employing a medium of the following formula:
soopic powder having a potency of 258 units/mg.
KHzPOi ____________________________ __g__ 1.5 45
Example 8
MgsOr'lHzO
g
Ammonium tartrate ________________ .._g__
Glucose _
CaCO:
FeSO4-7H2O
0.5
6
Employing a tap-water medium made up with
the following percentages of ingredients:
2
5.0
NaNOa
50
_________________________ __ trace
g
20
NazSOa
g
1.5
Distilled water ___________________ __liter_..
1
KH2P04
MgSOr'lI-IaO
_
0.6
0.15
0.05
0.5
in place of the medium in Example 1, and under
Corn steep liquor ______________________ __ 3.0
the same conditions, a maximum potency of 24 55 Brown sugar
1.0
units/ml. is developed in 6 days, the pH of the
NazSOa
_ 0.1
culture ?ltrate being '7.6. (Production of peni
in place of the medium in Example 1, and under
cillin in this “synthetic" medium compares favor
the
same conditions, a maximum potency of 44
ably with that in a regular deep-culture medium
units/ml.
is developed in 5 days, the pH of the
containing corn steep liquor which has incubated 60
culture ?ltrate being 8.1. (With the sodium sul
under the same conditions.)
?te omitted from this formula, and under the
Example 4
same conditions, a maximum potency of 36
units/ml. is developed in 5 days, the pH of the
With the sodium'sul?te and brown sugar in
the formula of Example 1 respectively increased 65 culture ?ltrate being 7.6.)
Example 9
to 1.5 g. and 20 g., and under the same condi
tions, a maximum potency of 48 units/ml. is de
Employing a medium of the following formula:
veioped in 6 days, the pH of the culture ?ltrate
being 8.5.
Example 5
CaCOs
KHzPOt
g
MgSOv'lHzO
_______________________
__g__
70
1.5
0.5
NaNOs
.._.._
g
6.0
With the sodium sul?te in the formula of Ex
CaCO:
___
g
5.0
ample 1 increased to 1.5 g., and 20 g. glucose in
Glucose ..__
g
20.0
cluded in place of-the brown sugar, and under
NazSOa
g
3.0
the same conditions, a maximum potency of 36
units/ml. is developed in 6 days, the pH of the 'll Tap water _______________________ __liter__. ‘ 1
amazon
5
6
in place or the medium in Example 1, and under
Example 15
The following ingredients:
the same conditions, a maximumv potency of 24
units/ml. is developed in 3 days, the pH of the
culture ?ltrate being 8.0. (With the sodium sul
Pounds
fite omitted from this formula, and under the 5 KH2P°4
__
8.8
same conditions, a maximum potency of 12
MgSOs?HzO ......................... .._
2.9
units/ml. is developed in 5 days, the pH of the
NaNO;
35
culture ?ltrate being 7.8. With the sodium sul
NaaSOs
11.’!
?te in this formula reduced to 0.5 8.. and under
Brown sugar ________________________ __ 117
the same conditions, a maximum potency of 20 10 CaCOs
29
units/ml. is developed in 5 days, the pH of the
are
dissolved
in
700
gals.
tap
water
in
a
l200-gal.
culture ?ltrate being 8.0; and with the sodium
tank equipped with a stirrer and a sparger for
sul?te in this formula increased to 8.0 g., and
under the same conditions, there is no change
introduction of air, 100 gals. water is added; the
in the maximum potency or time oidevelopment, 15 medium is sterilized; an inoculum of Penic?lium
notatum is added; and the medium is incubated at
but the pH 01’ the culture ?ltrate is 9+.)
25-26° C. while maintained under an air pres
Example 10
sure of about 15 lbs., with air passing into the
medium through the sparger. The tank is har
With the brown sugar in the formula of Exam
vested after 61/,» days incubation, the culture ?l
pie 1 increased to 20 g., and the sodium sul?te 20 trate
obtained having a potency of 36 units/m1.
replaced by 2 g. sodium thiosulfate, and under the
In the production of sodium-penicillin from
same conditions, a satisfactory yield of penicillin
this culture ?ltrate in the manner detailed here
is obtained.
inbefore, the emulsion resulting on extraction
Example 11
with amyl acetate is easier to resolve than the
Employing a medium of the following formula: 25 corresponding emulsion formed where corn steep
liquor has been included in the medium, and the
NaNOa ______________________________ _-g__
6
product (sodium-penicillin) dries in a different
KH2P04 -___
--g
1.5
}manner, yielding a ?u?ler and lighter powder.
MgSOr'IHzO ________________________ __g__ 0.5
This sodium-penicillin has been further puri
Brown sugar ________________________ _-g__ 20 30 ?ed, and obtained in pure crystalline form. Its
Na2S203'5H2O _______________________ “5--
4
Tap water ________________________ __llter__
1
formula, C14H1oO4N2SNa, and rotation, +290° (in
water at 23° C.) diiler from that of the pure crys
talline sodium-penicillin described in Winterstein
er and MacPhillamy application Serial No. 497,
in place of the medium in Example 1, and under
the same conditions, a potency of 24 units/ml. 35 ‘719, ?led August 6. 1943 and now abandoned
is developed in 4 days. This medium, incidentally,
(which was derived from the prior corn-steep
has the possible advantage of being perfectly
liquor containing submerged-culture media); but
clear.
the different products have approximately the
Example 12
same antibacterial potency.
40
The proportion of each of the ingredients in
Employing a tap-water medium made up with
cluded in the media of the foregoing examples
the following percentages of ingredients:
may be varied considerably without sacri?cing
MgSOv'II-IzO __________________________ __ 0.05
all of the advantage gained by inclusion of an
KNO;
inorganic, water-soluble sul?te-type compound.
0.6
45
Thus, variations of the NaNOa included from
about 0.3% to about 2.4%, of the CaCOa included
Ca:(PO4)-2
__..0.5
from about 0.1% to about 2%, of the MgSO4-7H2O
included from 0.05% to 0.2%, and of the brown
in place of the medium in Example 1, and under
sugar included from 1% to 6%, have been deter
the same conditions, a potency of 24 units/ml. 50 mined to be feasible (these, of course, not repre
is developed in 5 days.
senting the limits of the operative ranges); also
more concentrated media, e. g., one having twice
Example 13
NazSOa
_-._
..
..
0.2
Brown sugar
2.0
With the brown sugar in the formula of Ex
ample 1 increased to 20 8., and the sodium sul
?te replaced by 1 g. (or 3 g.) sodium metabisul
?te, and under the same conditions, a satisfactory
yield of penicillin is obtained.
Example 14
Employing a medium of the following formula:
the concentration of the medium described in Ex—
ample 4, may be employed.
55
-
The potassium, phosphorus, magnesium, cal
cium, and nitrogen supplied by the nutrient salts
may, of course, be supplied by salts other than
those speci?cally named hereinbefore; and one
or more of the (nutrient) metals may be sup
60 plied in whole or part by selection of the ap
propriate inorganic, water-soluble, sul?te-type
compound, inter alia, magnesium sul?te and po
tassium sul?te.
The media utilizable in the practice of this in
KHsPO4
g
‘1.5
MgSOr-‘ZHzO _______________________ .._g__ 0.5 65 vention may include (in addition to or in place of
NaNOz
_.._-
g__
CaCO:
2
Glucose __
g )
NazsOa
..-_-
-_-
6
g
5
ingredients mentioned hereinbefore) one or more
20
of the following, inter alia: urea, glycerol, mo
lasses, sucrose, starch, and various other pure
2
ZnSO4-7Hz0 _______________________ __g__ .001
FeSOrVHzO __________________________ __
ace
Distilled water ___________________ __liter__
1
in place of the medium in Example 1, and under
the same conditions, a satisfactory yield of peni
cillln is obtained.
'
or impure carbohydrates, soy-bean-eake meal,
70 cottonseed meal, yeast extract, wheat germ, whis
key slop, casein hydrolysate, tryptone, beef
heart-infusion broth, and Penicillium autolysate.
Preferably, the ingredients selected are those
which will provide wholly synthetic media.
75
The production of penicillin by submerged cul
2,437,918
ture in the synthetic liquid nutrient media of‘
this invention is enhanced by the inclusion or a
trace to about 0.01 % of zinc sulfate (or equivalent
' amount of other zinc salt).
Also, if a wholly syn
thetic liquid nutrient medium is made with dis
tilled water and a pure carbohydrate, a trace of
iron (desirably in the form of ferrous sulfate)
mg a proportion of sul?te radical to medium of
about 0.02% to about 0.65%, and incubatingthe
inoculated medium.
'
3. The method of producing penicillin which
comprises growing a penicillin-forming mold in
submerged culture in a synthetic liquid nutrient
medium including an ingredient of the group con
sisting of inorganic, water-soluble sul?tes, bisul
should be included.
?tes, metabisul?tes, hyposul?tes, and thiosul
The methods and media of this invention are
fates, which ingredient is included in amount
utillzable for- the production of penicillin or re 10 capable
of yielding a proportion of sul?te radical
lated antibiotics by the growth of (suitable
to
medium
of about 0.02% to about 0.65%.
strains) of antibiotic-forming molds other than ,
4. The method of producing penicillin which
Penicillium notatum, inter alia Penicillium chrys
comprises growing a penicillin-forming mold in
ogcnum and Aspergillus ?avus; and the term
submerged culture in a synthetic liquid .nutrient
15
“penicillin," as generically employed in the claims,
medium including an amount of sodium sul?te
is accordingly to be understood as embracing such
capable of yielding a proportion of sul?te radical
related antibiotics.
to medium of about 0.02% to about 0.65%.
The invention may be variously otherwise em
ROBERT B. McCORMACK.
bodied within the scope of the appended claims.
20
I claim:
REFERENCES CITED
1. The method of producing penicillin which
The following references are of record in the
comprises inoculating with a penicillin-forming
?le of this patent:
mold a synthetic liquid nutrient medium essen
tially including nutrient salts, a carbohydrate
UNITED STATES PATENTS
assimilable by the mold and a member of the 25
Name
Date
Number
group consisting of inorganic, water-soluble sul
?tes, bisul?tes, metabisul?tes, hyposulfites, and
thiosulfates, the last ingredient being included in
1,044,615
1,582,408
2,179,203
2,346,011
Willcox __________ __ Nov. 19, 1912
Legg _____________ __ Apr. 27, 1926
Stein _____________ __ Nov. 7, 1939
amount capable of yielding a proportion of sul?te
Damm ____________ .._ Apr. 4, 1944
radical to medium of about 0.02% to about 0.65%, 30
and incubating the inoculated medium.
OTHER. REFERENCES
2. The method of producing penicillin which
‘Foster et 211.: “Jr. of-Bacteriology," Nov, 1943,
comprises inoculating with a penicillin-forming
page 433.
mold a synthetic liquid nutrient medium includ
Summary of discussion at the Penicillin Tech
ing an ingredient of the group consisting of inor
nical
Meeting, New York, January 21-22, 1944,
ganic, water-soluble sulfites, bisulfltes, metabi
page 7.
sul?tes, hyposul?tes, and thiosulfates, which in
Chemical Abstracts: 361613 (3)‘, (1942).
gredient is included in amount capable of yield
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