Process for enzymatic deacylation of antibiotics

This invention relates to two new antibiotics, desacetyl 890A.sub.1 and desacetyl 890A.sub.3, active against both gram-positive and gram-negative bacteria, which are produced by treating 890A.sub.1 and 890A.sub.3, respectively, with an N-acetylthienamycin amidohydrolase produced by a soil microorganism isolated by enrichment techniques. This invention also relates to the process whereby N-acetylated structures of the thienamycin class of antibiotics such as N-acetyl thienamycin can be enzymatically deacetylated.

BACKGROUND OF THE INVENTION 
The discovery of the remarkable antibiotic properties of penicillin 
stimulated great interest in this field which has resulted in the finding 
of many other valuable antibiotic substances such as: other penicillins, 
streptomycin, bacitracin, tetracyclines, chloramphenicol, erythromycins 
and the like. In general, the antibacterial activity of each of these 
antibiotics does not include certain clinically important pathogenic 
bacteria. For example, some are principally active against only 
gram-positive types of bacteria. Acquired resistance over the course of 
widespread use of existing antibiotics in the treatment of bacterial 
infection has caused a serious resistance problem to arise. 
Accordingly, the deficiencies of the known antibiotics have stimulated 
further research to find other antibiotics which will be active against a 
wider range of pathogens as well as resistant strains of particular 
microorganisms. 
SUMMARY OF THE INVENTION 
This invention relates to two new antibiotic agents. More particularly, it 
is concerned with the new antibiotic substances, herein called desacetyl 
890A.sub.1 and desacetyl 890A.sub.3. The invention encompasses the 
antibiotics in dilute forms, as crude concentrates and in pure forms. 
It is an object of the present invention to provide the new and useful 
antibiotics which are highly effective in inhibiting the growth of various 
gram-negative and gram-positive microorganisms. Another object is to 
provide a process for the preparation of these novel antibiotic substances 
by the enzymatic deacetylation of the compounds 890A.sub.1 and 890A.sub.3. 
A further object of the present invention is to provide a process for the 
deacetylation of N-acetylthienamycin. Other objects will be apparent from 
the detailed description of this invention hereinafter provided. 
The novel antibiotic substances of the present invention are produced by 
hydrolyzing the N-acetyl group of 890A.sub.1 and 890A.sub.3 using an 
amidohydrolase capable of hydrolyzing the N-acetyl group. A convenient 
source of an amidohydrolase with this capability is amidohydrolase 
producing strains of the microorganism Protaminobacter ruber. The 
particular enzyme produced by Protaminobacter ruber is N-acetylthienamycin 
amidohydrolase, a member of the sub-group of enzymes designated B.C. 3.5.1 
according to the recommended enzyme nomenclature of the International 
Union of Pure and Applied Chemistry and the International Union of 
Biochemistry. 
The microorganism capable of carrying out the deacetylation process was 
isolated from a soil sample and, based upon taxonomic studies, was 
identified as belonging to the species Protaminobacter ruber and has been 
designated MB-3528 in the culture collections of MERCK & CO., Inc., 
Rahway, N.J. A culture thereof has been placed on unrestricted permanent 
deposit with the culture collection of the Northern Regional Research 
Laboratories, Northern Utilization Research and Development Division, 
Agricultural Research Service, U.S. Department of Agriculture, Peoria, 
Ill., and has been assigned accession No. NRRL B-8143. 
The morphological and cultural characteristics of Protaminobacter ruber 
NRRL B-8143 as well as carbon and nitrogen utilization and biochemical 
reactions are as follows: 
Morphology--Cells are rod-shaped with rounded ends, 0.9-1.2.times.2.3-4.6 
microns, occurring singly or in parts. Twenty-four and forty-eight hour 
cells stain gram-negative with a granular appearance. The granules, 
especially the polar granules, stain black with Sudan Black B. Cells are 
motile at 28.degree. C., but motility is questionable at 37.degree. C. 
Cultural Characteristics--Nutrient agar colonies are at first thin, 
punctiform, semi-transparent and colorless, the becoming low convex, 
opaque, smooth, edge entire, somewhat dry in consistency and pigmented 
rose to rose-red. 
Nutrient broth cultures are uniformly turbid with no pellicle. 
Pigment production is not dependent on light or temperatures tested 
(28.degree. C. and 37.degree. C.). Pigment is soluble in acetone but 
insoluble in water or chloroform. 
Growth on nutrient agar and brain-heart infusion agar under aerobic 
conditions is somewhat slow but good at 28.degree. C.; growth is moderate 
to good but slower at 37.degree. C.; there is not growth at 50.degree. C. 
UTILIZATION OF CARBON AND NITROGEN SOURCES 
Using a basal salts medium with ammonium sulfate as nitrogen source, growth 
is good with arabinose, moderate with xylose, and poor with dextrose, 
fructose, mannose, rhamnose, lactose, maltose, sucrose, raffinose, 
cellulose, inositol and mannitol. 
N-acetylethanolamine can be utilized as the sole carbon and nitrogen 
source. 
No acid or gas is produced from dextrose or lactose in OF Basal Medium 
(Difco Laboratories, Detroit, Mich.) under aerobic or anaerobic 
conditions. 
BIOCHEMICAL REACTIONS 
The biochemical reactions are based on standard methods as described in 
Manual of Microbiological Methods edited by the Society of American 
Bacteriologists, McGrawHill Book Co., New York, 1957. 
Catalase--positive 
Oxidase--negative 
Starch not hydrolyzed 
Casein not hydrolyzed 
Gelatin not liquefied 
Litmus milk unchanged in consistency but becomes slightly alkaline after 7 
days. 
Indol--negative 
H.sub.2 S--negative 
Nitrates not reduced 
Urease--positive 
Lysine and ornithine decarboxylase--negative 
N-Acetylthienamycin, 890A.sub.1 and 890A.sub.3 are the terms applied to 
isomers of the antibiotic having the structure: 
##STR1## 
N-Acetylthienamycin, its description and process of production are set 
forth in the application of Kahan et al., U.S. Ser. No. 634,301, filed 
Nov. 21, 1975 now abandoned, which is herein incorporated by reference. 
890A.sub.1 and 890A.sub.3, their description and processes of production 
are set forth in the application of Cassidy et al., U.S. Ser. No. 634,300, 
filed, filed on Nov. 21, 1975 now abandoned which is herein incorporated 
by reference. 
The novel antibiotics of the present invention, desacetyl 890A.sub.1 and 
desacetyl 890A.sub.3 are isomers of the structural formula: 
##STR2## 
and are prepared by enzymatic hydrolysis of 890A.sub.1 and 890A.sub.3, 
respectively using an amidohydrolase present in species of genus 
Protaminobacter. 
The novel process of the present invention relates to the cleavage of the 
N-acetyl group of the compounds of the structure: 
##STR3## 
which comprises intimately contacting said compounds with an 
amidohydrolase capable of hydrolyzing the N-acetyl group. More 
specifically the process of the present invention provides for the 
N-deacetylation of N-acetylthienamycin, 890A.sub.1 and 890A.sub.3 by 
intimately contacting said compounds with the amidohydrolase, 
N-acetylthienamycin amidohydrolase. 
An unexpected homology between N-acetylethanolamine and N-acetylthienamycin 
is set forth, whereby extracts of microorganisms with the hitherto 
undescribed enzyme, N-acetylethanolamine amidohydrolase, are in many cases 
able to hydrolyze N-acetylthienamycin. Furthermore, amidohydrolases 
capable of hydrolyzing N-acetylthienamycin are also found to be active in 
the conversion of antibiotics 890A.sub.1 and 890A.sub.3 to their novel 
desacetyl forms. Another novel aspect of the present invention relates to 
the process for obtaining such microorganisms, wherein the process 
comprises selecting the strains capable of using N-acetylethanolamine for 
growth and then testing these microorganisms for the presence of 
N-acetylthienamycin amidohydrolase. 
The antibiotic thienamycin, obtained by the novel N-deacetylation of 
N-acetylthienamycin is a useful antibiotic. Its description, method of 
production and utility are set forth in the application of Kahan et al., 
U.S. Ser. No. 526,992, filed Nov. 25, 1974 U.S. Pat. No. 3,950,357 which 
is incorporated herein by reference. The antibiotics desacetyl 890A.sub.1 
and 890A.sub.3 are novel and useful antibiotics. 
One aspect of the present invention is the novel deacetylation of 
N-acetylthienamycin. There are two sources of N-acetylthienamycin. 
N-acetylthienamycin is prepared by the fermentation of broth with the 
microorganism Streptomyces cattleya NRRL 8057. This microorganism also 
produces thienamycin which may be chemically N-acetylated. 
Based upon extensive taxonomic studies, Streptomyces cattleya, isolated 
from a soil sample, was identified as an actinomycete and has been 
designated MA-4297 in the culture collection of MERCK & CO., Inc., Rahway, 
N.J. A culture thereof has been placed on unrestricted permanent deposit 
with the culture collection of the Northern Regional Research 
Laboratories, Northern Utilization Research and Development Division, 
Agricultural Research Service, U.S. Department of Agriculture, Peoria, 
Ill., and has been assigned accession No. NRRL 8057. 
The classification keys for the genus Streptomyces and the culture 
descriptions of Streptomyces species found in Bergey's Manual of 
Determinative Bacteriology (7th Edition, 1957) and in The Actinomycetes, 
Vol. II (1961) by S. A. Waksman and in "Cooperative Descriptions of Type 
Cultures of Streptomyces" by E. B. Shirling and D. Gottlieb, International 
Journal of Systematic Bacteriology, 18, 69-189 (1968), 18, 279-392 (1968), 
19, 391-512 (1969) and 22, 265-394 (1972) were searched for a Streptomyces 
species having morphological and cultural characteristics similar to those 
of MA-4297. In these aforementioned classical references, no Streptomyces 
species is described to have the orchid pigmentation of the aerial 
mycelium, the morphological characteristics and the absence of diffusible 
pigment which together comprise distinctive characteristics of MA-4297. 
These considerations made the assignment of a new Streptomyces species 
justified and necessary. 
The morphological and cultural characteristics of Streptomyces cattleya are 
set forth in the following table. 
Morphology--Sporophores are compact spirals occurring as side and terminal 
branches on aerial mycelium. Spores are ellipsoidal to cylindrical in 
shape, 0.9.mu..times.1.2.mu. in size, occurring in chains of more than 10. 
CULTURAL 
Tomato paste-oatmeal agar 
Vegetative growth--Reverse-tan, flat, spreading: 
Aerial mycelium--Orchid (10 gc) mixed with white; 
Soluble pigment--None. 
Czapek Dox agar (sucrose nitrate agar) 
Vegetative growth--Colorless, flat, spreading; 
Aerial mycelium--Sparse, pinkish white; 
Soluble pigment--None. 
Egg albumin agar 
Vegetative growth--Tan with grayed-orchid case, flat, spreading; 
Aerial mycelium--Orchid (10 gc) mixed with lighter shades of orchid and 
some white; 
Soluble pigment--None. 
Glycerol asparagine agar 
Vegetative growth--Reverse-tan with gray-pink cast, flat, spreading; 
Aerial mycelium--Orchid (10 gc) mixed with some white; 
Soluble pigment--None. 
Yeast extract-glucose+salts agar 
Vegetative growth--Tan with grayed pink cast; 
Aerial mycelium--Orchid (10 gc) mixed with pinkish-white; 
Soluble pigment--None. 
Yeast extract-malt extract agar 
Vegetative growth--Tan; 
Aerial mycelium--Orchid (10 gc) mixed with pinkish-white; 
Soluble pigment--None. 
Peptone-iron-yeast extract agar 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--Slight browning of medium; 
Melanin--Negative; 
H.sub.2 S production--Negative. 
Nutrient agar 
Vegetative growth--Light tan; 
Aerial mycelium--None; 
Soluble pigment--None. 
Nutrient starch agar 
Vegetative growth--Cream to tan; 
Aerial mycelium--None; 
Soluble pigement--None; 
Hydrolysis of starch--Moderate. 
Nutrient gelatin agar 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction of gelatin--moderate. 
Gelatin stabs 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction of gelatin--Moderate. 
Potato plug 
Vegetative growth--Moderate, tan; 
Aerial mycelium--Sparse, grayish-pinkish-white; 
Soluble pigment--None. 
Loeffler's Blood serum 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction--None. 
Skim milk agar 
Vegetative growth--Tan; 
Aerial mycelium--Sparse, whitish; 
Soluble pigment--Slight browning of medium; 
Hydrolysis of casein--Positive. 
Litmus milk 
Vegetative growth--Tan to brown; 
Aerial mycelium-None; 
Color--No soluble pigment, litmus indicator becoming bluish; 
Coagulation and/or peptonization--Partial peptonization, becoming alkaline. 
Skim milk 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--None; 
Coagulation and/or peptonization--Partial peptonization, becoming alkaline. 
Tyrosine agar 
Vegetative growth--Tan; 
Aerial mycelium--Mixture of orchid (10 gc) and white; 
Soluble pigment--None; 
Decomposition of tyrosine--positive. 
All of the readings reported above were taken after three weeks incubation 
at 28.degree. C. unless noted otherwise. The pH of the media used in these 
studies was approximately neutral, namely, pH 6.8-7.2. The color 
designations used in the description are in accordance with the 
definitions of the Color Harmony Manual, 4th Edition (1956), Container 
Corporation of America, Chicago, Illinois. 
Streptomyces cattleya was also tested for its ability to utilize or 
assimilate various carbohydrates. For this purpose, the microorganism was 
grown on basal synthetic medium (Pridham and Gottlieb) containing 1% of 
the carbohydrate at 28.degree. C. for three weeks. The pH of the media 
employed in the study was approximately neutral (6.8-7.2). Table I shows 
the utilization of these carbohydrate sources by Streptomyces cattleya: 
+indicating good growth, .+-.poor growth, and -no growth on the particular 
carbohydrate. 
TABLE I 
______________________________________ 
Glucose + Maltose .+-. 
Arabinose - Mannitol + 
Cellulose - Mannose .+-. 
Fructose .+-. Raffinose - 
Inositol - Rhamnose - 
Lactose - Sucrose .+-. 
Xylose .+-. 
______________________________________ 
The amount of growth with change in temperature, the oxygen requirement and 
the effect on nitrate by the microorganism is as follows: 
Temperature range (Yeast extract-glucose+salts agar); 
28.degree. C.--Good 
37.degree. C.--Moderate 
50.degree. C.--No growth 
Oxygen requirement (Stab culture in yeast extract-glucose+salts agar); 
Aerobic 
Nitrate reduction--Positive. 
It is to be understood that the production of thienamycin is not limited to 
the organism Streptomyces cattleya or to organisms fully answering the 
above growth and microscopic characteristics which are given for 
illustrative purposes. In fact, it is desired and intended to include the 
use of mutants produced from the described organism by various means, such 
as X-radiation, ultraviolet radiation, nitrogen mustard, phage exposure 
and the like. 
Thienamycin is produced during the aerobic fermentation of suitable aqueous 
nutrient media under controlled conditions via the inoculation with the 
organism, Streptomyces cattleya. Aqueous media, such as those employed for 
the production of other antibiotics are suitable for producing 
thienamycin. Such media contain sources of carbon, nitrogen and inorganic 
salts assimilable by the microorganism. 
Another aspect of the present invention in the novel deacetylation of 
890A.sub.1 and 890A.sub.3. 890A.sub.1 and 890A.sub.3 are prepared by 
fermentation of broth with the microorganism Streptomyces flavogriseus 
NRRL 8139. The material 890A.sub.1 may also be prepared by fermentation of 
Streptomyces flavogriseus NRRL 8140. 
Based upon extensive taxonomic studies the strains of microorganisms were 
identified as belonging to the species Streptomyces flavogriseus and have 
been designated MA-4434a and MA-4600a in the culture collection of MERCK & 
CO., Inc., Rahway, N.J. A culture of each thereof has been placed on 
irrevocable permanent deposit with the culture collection of the Northern 
Regional Laboratories, Northern Utilization Research and Development 
Division, Agricultural Research Service, U.S. Department of Agriculture, 
Peoria, Ill., and have been assigned accession No. NRRL 8139 and 8140, 
respectively. 
Streptomyces flavogriseus NRRL 8139 produces both antibiotics 890A.sub.1 
and 890A.sub.3 which are isolated in substantially pure form from the 
fermentation broth. Streptomyces flavogriseus NRRL 8140 produces 
antibiotic 890A.sub.1 without any detectable amount of 890A.sub.3. 
The morphological and cultural characteristics of Streptomyces flavogriseus 
NRRL 8139 are set forth in the following table. 
Morphology--Sporophores are branching, straight to flexuous chains of 
spores, forming, tufts. Chains are more than 10 spores in length. Spores 
are spherical to oval-0.9.mu..times.1.2.mu.(970x). 
CULTURAL CHARACTERISTICS 
Oatmeal agar 
Vegetative growth--Reverse-yellowish tan, parchment-like growth; 
Aerial mycelium--Light gray edged with medium gray 
Soluble pigment--None. 
Czapek Dox agar (sucrose nitrate agar) 
Vegetative growth--Reverse-brown edged with dark brown; 
Aerial mycelium--Medium gray, velvety; 
Soluble pigment--Slight browning of medium. 
Egg albumin agar 
Vegetative growth--Reverse-yellowish tan edged with brown; 
Aerial mycelium--Medium gray mixed with yellowish gray (2 dc) and grayed 
yellow (2 db); 
Soluble pigment--Light yellowish tan. 
Glycerol asparagine agar 
Vegetative growth--Reverse-yellowish tan, flat, spreading; 
Aerial mycelium--Velvety, light gray with a strong yellowish tone to gray 
(2 dc); 
Soluble pigment--None. 
Inorganic salts-starch agar 
Vegetative growth--Reverse-brown; 
Aerial mycelium--Medium gray, velvety; 
Soluble pigment--Light yellowish-tan. 
Yeast extract-dextrose+salts agar 
Vegetative growth--Reverse-brown edged with very dark brown; 
Aerial mycelium--Dark gray mixed with a light gray, velvety; 
Soluble pigment--None. 
Yeast extract-malt extract agar 
Vegetative growth--Reverse-dark brown; 
Aerial mycelium--Dark grey, velvety; 
Soluble pigment--None. 
Skim milk agar 
Vegetative growth--Tan; 
Aerial mycelium--Sparse, grayish; 
Soluble pigment--Slight browning of medium; 
Hydrolysis of casein--Good. 
Litmus milk 
Vegetative growth--Moderate growth ring, dark tan; 
Aerial mycelium--None; 
Color--Purple; 
Coagulation and/or peptonization--Complete peptonization; becoming 
alkaline, pH 8.2. 
Skim milk 
Vegetative growth--Moderate growth ring, tan; 
Aerial mycelium--None; 
Soluble pigment--Tan; 
Coagulation and/or peptonization--Complete peptonization; becoming 
alkaline, pH 8.0. 
Tyrosine agar 
Vegetative growth--Reverse-dark brown; 
Aerial mycelium--Dark gray; 
Soluble pigment--Slight browing of medium; 
Decomposition of tyrosine--None. 
Peptone-iron-yeast extract agar 
Vegetative growth--Tan; 
Aerial mycelium--Sparse, grayish; 
soluble pigment--None; 
Melanin--None; 
H.sub.2 S production--None. 
Nutrient agar 
Vegetative growth--Reverse-light grayish brown edged with darker 
gray-brown; 
Aerial mycelium--Light gray edged with dark gray; 
Soluble pigment--None. 
Nutrient starch agar 
Vegetative growth--Tan edged with gray 
Aerial mycelium--Medium gray edged with dark gray; 
Soluble pigment--None; 
Hydrolysis of starch--Good 
Nutrient gelatin agar 
Vegetative growth--Colorless edged with dark gray; 
Aerial mycelium--Grayish-white 
Soluble pigment--None; 
Liquefaction of gelatin--Good. 
Potato plug 
Vegetative growth--Good growth, heavily wrinkled; 
Aerial mycelium--Gray to greenish-gray; 
Soluble pigment--Slight browning of medium. 
Loeffler's Blood serum 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction--None. 
Gelatin stabs 
Vegetative growth--Cream-colored; 
Aerial mycolium--None; 
Soluble pigment--None; 
Liquefaction of gelatin--Good. 
All of the readings reported above were taken after three weeks incubation 
at 28.degree. C. unless noted otherwise. The pH of the media used in these 
studies was approximately neutral, namely, pH 6.8-7.2. The color 
designations used in the description are in accordance with the 
definitions of the Color Harmony Manual, 4th Edition (1958), Container 
Corporation of America, Chicago, Illinois. 
Streptomyces flavogriseus NRRL 8139 was also tested for its ability to 
utilize or assimilate various carbohydrates. For this purpose, the 
microorganism was grown on basal synthetic medium (Pridham and Cottlieb) 
containing 1% of the carbohydrate at 28.degree. C. for three weeks. The pH 
of the media employed in the study was approximately neutral (6.8-7.2). 
Table II shows the utilization of these carbohydrate sources by 
Streptomyces flavouriseus NRRL 8139, + indicating good growth, .+-. poor 
growth, and - no growth on the particular carbohydrate. 
TABLE II 
______________________________________ 
Glucose + Maltose + 
Arabinose + Mannitol + 
Cellulose - Mannose + 
Fructose + Raffinose - 
Inositol - Rhamnose + 
Lactose + Sucrose .+-. 
Xylose + 
______________________________________ 
The amount of growth with change in temperature and the oxygen requirement 
by the microorganism is as follows: 
Temperature range (Yeast extract-dextrose+salts agar); 
28.degree. C.--Good 
37.degree. C.--Good vegetative growth; no aerial hyphae 
50.degree. C.--No growth 
Oxygen requirement (Stab culture in yeast extract-dextrose+salts agar); 
Aerobic 
The morphological and cultural characteristics of Streptomyces flavogriseus 
NRRL 8140 are set forth in the following table. 
Morphology--Sporophores are branching, straight to flexuous chains of 
spores, forming tufts. Chains are more than 10 spores in length. Spores 
are spherical to oval--0.9.mu..times.1.2.mu. (970x). 
CULTURAL CHARACTERISTICS 
Oatmeal agar 
Vegetative growth--Reverse-yellowish tan edged with dark brown; 
Aerial mycelium--Light gray edged with medium gray; 
Soluble pigment--None. 
Czapek Dox agar (sucrose nitrate agar) 
Vegetative growth--Reverse-brown edged with dark brown; 
Aerial mycelium--Medium gray, velvety; 
Soluble pigment--None. 
Egg albumin agar 
Vegetative growth--Reverse-grayish tan with sections of strong yellow tan; 
Aerial mycelium--Sections of medium gray, grayish white and yellowish g. y 
(2dc); Soluble pigment--Very light tan. 
Glycerol asparagine agar 
Vegetative growth--Yellowish tan; 
Aerial mycelium--Sparse, grayish; 
Soluble pigment--None. 
Inorganic salts-starch agar 
Vegetative growth--Reverse-grayish cream; 
Aerial mycelium--Medium gray, velvety; 
Soluble pigment--None. Yeast extract-dextrose+salts agar 
Vegetative growth--Reverse-dark brown; 
Aerial mycelium--Dark gray mixed with a light gray, velvety; 
Soluble pigment--None. 
Yeast extract-malt extract agar 
Vegetative growth--Reverse-dark brown; 
Aerial mycelium--Dark gray, velvety; 
Soluble pigment--None. 
Peptone-iron-yeast extract agar 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--None; 
Melanin--None; 
H.sub.2 S production--None. 
Nutrient agar 
Vegetative growth--Light tan; 
Aerial mycelium--None. 
Soluble pigment--None. 
Nutrient starch agar 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Hydrolysis of starch--Good. 
Nutrient gelatin agar 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction of gelatin--Good. 
Gelatin stabs 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction of gelatin--Complete. 
Skim milk agar 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--None; 
Hydrolysis of casein--Good. 
Litmus milk 
Vegetative growth--Tan growth ring 
Aerial mycelium--None; 
Color--Brownish purple; 
Coagulation and/or peptonization--complete peptonization, becoming 
alkaline, pH 8.0. 
Skim milk 
Vegetative growth--Tan, moderate growth ring; 
Aerial mycelium--None; 
Soluble pigment--Light brown; 
Coagulation and/or peptonization--Complete peptonization, becoming alkaline 
pH 8.5. 
Potato plug 
Vegetative growth--Good, tan colored; 
Aerial mycelium--Very sparse, whitish; 
Soluble pigment--None. 
Loeffler's Blood serum 
Vegetative growth--Cream-colored; 
Aerial mycelium--None; 
Soluble pigment--None; 
Liquefaction--None. 
Tyrosine agar 
Vegetative growth--Tan; 
Aerial mycelium--None; 
Soluble pigment--Slight browning of medium; 
Decomposition of tyrosine--Very slight. 
All of the readings reported above were taken after three weeks incubation 
at 28.degree. C. unless noted otherwise. The pH of the media used in these 
studies was approximately neutral, namely pH 6.8-7.2. The color 
designations used in the description are in accordance with the 
definitions of the Color Harmony Manual, 4th Edition, 1958), Container 
Corporation of America, Chicago, Illinois. 
Streptomyces flavogriseus NRRL 8140 was also tested for its ability to 
utilize or assimilate various carbohydrates. For this purpose, the 
microorganism was grown on basal synthetic medium (Pridham and Gottlieb) 
containing 1% of the carbohydrate at 28.degree. C. for three weeks. The pH 
of the media employed in the study was approximately neutral (6.8-7.2) 
Table III shows the utilization of these carbohydrate sources by 
Streptomyces flavogriscus NRRL 8140+indicating good growth, .+-.poor 
growth, and -no growth on the particular carbohydrate. 
TABLE III 
______________________________________ 
Glucose + Maltose + 
Arabinose + Mannitol + 
Cellulose - Mannose + 
Fructose + Raffinose .+-. 
Inositol .+-. Rhamnose + 
Lactose + Sucrose .+-. 
Xylose + 
______________________________________ 
The amount of growth with change in temperature and the oxygen requirement 
by the microorganism is as follows: 
Temperature range (Yeast extract-dextrose+salts agar); 
28.degree. C.--Good 
37.degree. C.--Moderate vegetative growth; no aerial hyphae 
50.degree. C.--No growth 
Oxygen requirement (Stab culture in yeast extract-dextrose+salts agar); 
Aerobic. 
PHYSICAL AND CHEMICAL PROPERTIES OF ANTIBIOTICS 890A.sub.1 and 890A.sub.3 
Properties of Antibiotic 890A.sub.1 
Antibiotic 890A.sub.1 is an acidic substance which moves toward the 
positive pole on electrophoresis at neutral pH. 
The sodium salt of antibiotic 890A.sub.1 is a white powder as lyophilized 
from aqueous solution, and is very soluble in water. 
The ultraviolet absorbance spectrum has a .lambda.max. at 299.5 nm and a 
.lambda.min. at 242 nm. The E% at 300 nm of a solution of the sodium salt 
of antibiotic 890A.sub.1 in water at neutral pH is 208 for a pure sample; 
the ratio of absorbance values at 300 nm and 245 nm is 4.25 and the ratio 
A.sub.300 /A.sub.210 is 1.41. More than 92% of the absorption at 300 nm 
may be eliminated by reaction with hydroxylamine, and a similar decrease 
is observed upon reaction with cysteine. 
The circular dichroism spectrum of 890A.sub.1 displays a positive maximum 
at 290.5 nm with a specific ellipticity of 5270 degree-ml. per 
decimeter-gram, a point of zero ellipticity at 250 nm, and a negative 
minimum at 214 nm, with specific ellipticity of -10,910 degree-ml. per 
decimeter-gram. 
The following table lists the 100 MHz-NMR signals for 890A.sub.1 sodium 
salt in D.sub.2 O relative to the internal standard, sodium 
2,2-dimethyl-2-silapentane-5-sulfonate, hereinafter referred to as DSS; 
chemical shifts are given in ppm and coupling constants in Hz; the 
apparent multiplicities are indicated. 
1.35 (d, J=6.5); 1.98 (s); 3.63 (d of d, J=5.2 and J=9.8); .about.4.02-4.26 
(m); 3.18 (d of d, J=.about.12 and J=10); 3.41 (t, J=6); 2.97 (d of t, 
J=3.5 and J=6). 
PROPERTIES OF ANTIBIOTIC 890A.sub.3 
Antibiotic 890.sub.3 is an acidic substance which moves to the positive 
pole on electrophoresis at neutral pH. 
The sodium salt is a white powder when lyophilized from aqueous solution. 
The ultraviolet absorbance spectrum has a maximum at 300.5 nm and a minimum 
at 243 nm. The E% at 300 nm of a solution of the sodium salt of antibiotic 
890A.sub.3 in water at neutral pH is 375 for a pure sample; the ratio of 
absorbances at 300 nm and 245 nm is 3.54. More than 90% of the absorption 
at 300 nm may be eliminated by reaction with hydroxylamine; and a similar 
decrease is observed upon reaction with cysteine. 
The circular dichroism spectrum of 890A.sub.3 has a positive maximum at 294 
nm with a specific ellipticity of 9127 degrees-ml. per decimeter-gram, a 
point of zero ellipticity at 249 nm, and a specific ellipticity of -15,053 
degrees-ml. per decimeter-gram at 220 nm. 
The following table lists the 100 MHz-NMR signals for 890A.sub.3 sodium 
salt in D.sub.2 O relative to the internal standard DSS; chemical shifts 
are given in ppm and coupling constants in Hz; the apparent multiplicities 
are indicated. 
1.29 (d, J=6.5); 1.98 (s); 3.42 (d of d, J=5 and J=2.4); .about.4.01-4.28 
(m); 3.14 (d of d, J=5 and J=9); 3.39 (t, J=6.5); 2.92 (d of t, J=.about.4 
and J=6). 
MASS SPECTRAL ANALYSIS OF 890A.sub.1 and 890A.sub.3 
The mass spectral data for 890A.sub.1 and 890A.sub.3 are obtained on 
trimethylsilyl derivatives prepared from ammonium salts of the antibiotics 
with bis-trimethylsilyltrifluoro acetamide in dimethyl formamide. 
Conversions of sodium salts of the antibiotics to the ammonium salts is 
carried out by using the ammonium salt of an acidic ion exchange resin. 
Trimethyl-silylation of 890A.sub.1 and 390A.sub.3 results in three 
different derivatives: a di- and a tri-trimethylsilyl derivative (M.W.s 
458 and 530, respectively) and a small amount of a tetra-trimethylsilyl 
derivative of a hydrolysed product (M.W. 620) wherein the .beta.-lactam 
ring is open. 
The values of the most important mass spectral fragment are given below: 
di-trimethylsilyl derivative; 443.1495; 301.1034; 300.0962; 241.0590 and 
86.0610. 
tri-trimethylsilyl derivative: 515.1931; 373.1422 and 158.1000. 
tetra-trimethylsilyl derivative (only low-resolution signal observed): 620 
and 605. 
Antibiotics 890A.sub.1 and 890A.sub.3 are isomers having a molecular 
structure as follows: 
##STR4## 
It is to be understood that the production of 890A.sub.1 and 890A.sub.3 is 
not limited to the organism, Streptomyces flavogriseus or to organisms 
fully answering the above growth and microscopic characteristics which are 
given for illustrative purposes. In fact, it is desired and intended to 
include the use of mutants produced from the described organism by various 
means, such as X-radiation, ultra-violet radiation, nitrogen mustard, 
phage exposure and the like. 
890A.sub.1 and 890A.sub.3 are produced during the aerobic fermentation, 
under controlled conditions, of suitable aqueous nutrient media inoculated 
with strains of the organism, Streptomyces flavogriseus. Aqueous media, 
such as those employed for the production of other antibiotics are 
suitable for producing 890A.sub.1 and 890A.sub.3. Such media contain 
sources of carbon, nitrogen and inorganic salts assimilable by the 
microorganism. 
Desacetyl 890A.sub.1 and desacetyl 890A.sub.3, the compounds of this 
invention are valuable antibiotics active against various gram-positive 
and gram-negtive bacteria and, accordingly, find utility in human and 
veterinary medicine. The compounds of this invention can be used as 
antibacterial drugs for treating infections caused by gram-positive or 
gram-negative bacteria, for example against susceptible strains of 
Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella 
pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The 
antibacterial materials of the invention may further be utilized as 
additives to animal feedingstuffs, for preserving foodstufs and as 
disinfectants. For example, they may be employed in aqueous compositions 
in concentrations ranging from 0.1 to 100 parts of antibiotic per million 
parts of solution or preferably in concentrations ranging from about 1 to 
about 10 parts of antibiotic per million parts of solution in order to 
destroy and inhibit the growth of harmful bacteria on medical and dental 
equipment and as bactericides in industrial applications, for example in 
water-based paints and in the white water of paper mills to inhibit the 
growth of deleterious bacteria. 
The antibiotics of this invention may be used in any one of a variety of 
pharmaceutical preparations as the sole active ingredient or in 
combination either with one or more other antibiotics or with one or more 
pharmacologically active substances. As an example of the former, an 
aminocyclitol antibiotic such as gentamicin may be coadministered in order 
to minimize any chance that resistant organisms will emerge. As an example 
of the latter, diphenoxylate and atropine may be combined in dosage forms 
intended for the therapy of gastroenteritis. The antibiotics may be 
employed in capsule form or as tablets, powders or liquid solutions or as 
suspensions or elixirs. They may be administered orally, topically, 
intravenously or intramuscularly. 
Tablets and capsules for oral administration may be in unit dose 
presentation form, and may contain conventional excipients such as binding 
agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or 
polyvinylpyrrolidone; fillers, for example, lactose, sugar, maizestarch, 
calcium phosphate, sorbitol or glycine; lubricants, for example, magnesium 
stearate, talc, polyethylene glycol, silica; disintegrants, for example, 
potato starch or acceptable wetting agents such as sodium lauryl sulphate. 
The tablets may be coated according to methods well known in the art. Oral 
liquid preparations may be in the form of aqueous or oily suspension, 
solution, emulsions, syrups, elixirs, etc. or may be presented as a dry 
product, for reconstitution with water or other suitable vehicles before 
use. Such liquid preparations may contain conventional additives such as 
suspending agents, for example, sorbitol syrup, methyl cellulose, 
glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl 
cellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying 
agents, for example lecithin, sorbitan monooleate or acacia; nonaqueous 
vehicles which may include edible oils, for example, almond oil, 
fractionated coconut oil, oily esters, propylene glycol, or ethyl alcohol; 
preservatives, for example methyl or propyl p-hydroxybenzoates or sorbic 
acid. Suppositories will contain conventional suppository bases, e.g. 
cocoa butter or other glyceride. 
Compositions for injection may be presented in unit dose form in ampules, 
or in multidose containers with an added preservative. The compositions 
may take such forms as suspensions, solutions, emulsions in oily or 
aqueous vehicles, and may contain formulatory agents such as suspending, 
stabilizing and/or dispersing agents. Alternatively, the active ingredient 
may be in powder form for reconstitution with a suitable vehicle, e.g. 
sterile, pyrogen-free water, before use. 
The compositions may also be prepared in suitable forms for absorption 
through the mucous membranes of the nose and throat or bronchial tissues 
and may conveniently take the form of powder or liquid sprays or 
inhalants, lozenges, throat paints, etc. For medication of the eyes or 
ears, the preparations may be presented as individual capsules, in liquid 
or semi-solid form, or may be used as drops etc. Topical application may 
be formulated in hydrophobic or hydrophilic bases as ointments, creams, 
lotions, paints, powders, etc. 
Also, in addition to a carrier, the instant compositions may include other 
ingredients such as stabilizers, binders, antioxidants, preservatives, 
lubricators, suspending agents, viscosity agents or flavoring agents and 
the like. 
In veterinary medicine, such as in the treatment of chickens, cows, sheep, 
pigs and the like, the compositions may, for example, be formulated as 
intramammary preparations in either long-acting or quick-release bases. 
The dosage to be administered depends to a large extent upon the condition 
of the subject being treated, the weight of the host and the type of 
infection, the route and frequency of administration, the parenteral route 
being preferred for generalized infections and the oral route for 
intestinal infections. 
In the treatment of bacterial infections in man, the compounds of this 
invention are administered orally or parenterally, in accordance with 
conventional procedures for antibiotic administration, in an amount of 
from about 2 to 600 mg./kg./day and preferably about 5 to 100 mg./kg./day 
in preferably divided dosage, e.g. three to four times a day. They may be 
administered in dosage units containing, for example, 25, 250, 330, 400 or 
1000 mg. of active ingredient with suitable physiologically acceptable 
carriers or excipients. The dosage units are in the form of liquid 
preparations such as solutions or suspensions or as solids in tablets or 
capsules. It will, of course, be understood that the optimum dose in ny 
given instance will depend upon the type and severity of infection to be 
treated, and that smaller doses will be employed for pediatric use, all of 
such adjustments being within the skill of the practitioner in the field. 
It is to be understood that the antibiotic thienamycin is also administered 
in the manner set forth above for the antibiotics desacetyl 890A.sub.1 and 
desacetyl 890A.sub.3. 
ASSAY PROCEDURES FOR N-ACETYL THIENAMYCIN 
I. Bioassay 
Assays of antibacterial activity are run according to the following 
disc-diffusion method using either Vibrio percolans ATCC 8461 or 
Staphylococcus aureus ATCC 6538P as tester organism. 
Plates containing Vibrio percolans ATCC 8461 are prepared as follows: 
A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml. 
of a sterilized medium containing 8 g./l. of Difco Nutrient Broth and 2 
g./l. of yeast extract in distilled water (hereinafter designated NBYE). 
The culture is incubated overnight on a rotary shaker at 28.degree. C. 
This culture is used to inoculate the surface of slants containing 1.5% 
agar in NBYE, and the inoculated slants are incubated overnight at 
28.degree. C. and then stored in a refrigerator. 
The refrigerated slants prepared from a single lyophilized culture are used 
for up to four weeks from their preparation, as follows: A loop of 
inoculum from the slant is dispersed in 50 ml. of NBYE contained in a 250 
ml. Erlenmeyer flask. The culture is incubated overnight on a rotary 
shaker at 28.degree. C., and is then diluted to a density giving 50% 
transmittance at 660 nm. A 33.2 ml. portion of this diluted culture is 
added to 1 liter of NBYE containing 15 g. of agar and maintained at 
46.degree. C. The inoculated agar-containing medium is poured into 
100.times.15 mm. plastic petri dishes, 5 ml. per dish, chilled, and 
maintained at 2.degree.-4.degree. C. for up to 5 days before using. 
Plates containing Staphylococcus aureus ATCC 6538P are prepared as follows: 
An overnight growth of the assay organism, Staphylococcus aureus ATCC 
6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient 
broth plus 0.2% yeast extract to a suspension having 55% transmittance at 
a wavelength of 660 nm. This suspension is added to Difco nutrient agar 
supplemented with 2.0 g./l. Difco yeast extract at 47.degree. C. to 
48.degree. C., to make a composition containing 33.2 ml. of the suspension 
per liter of agar. Five ml. of this suspension is poured into petri dishes 
of 85 mm. diameter, and these plates are chilled and held at 4.degree. C. 
until used (5 day maximum). 
Samples of antibiotic to be assayed are diluted to an appropriate 
concentration in phosphate buffer at ph 7. Filter paper discs, 1/4 or 1/2 
inch diameter, are dipped into the test solution and placed on the surface 
of the assay plate. The plates are incubated at 37.degree. C. overnight, 
and the zone of inhibition is measured as mm. diameter. The zone of 
inhibition measured in mm, determines relative potencies. 
II. Hydroxylamine-extinguishable absorbance 
The proportion of absorbance measured at 301 nm which can be attributed to 
the antibiotic content in impure samples is determined by the selective 
extinction of this absorbance (with concommitant inactivation of 
antibiotic activity) upon reaction with dilute hydroxylamine. 
Samples containing antibiotic to be tested are prepared in 0.01 M potassium 
phosphate buffer at pH 7 to have an initial A.sub.301 between 0.1 and 1.0. 
Freshly prepared, neutralized hydroxylamine (NH.sub.2 OH.HCl plus NaOH to 
a final pH of 7) is added to a final concentration of 10 mM, and the 
reaction is allowed to progress at room temperature for at least 30 
minutes. The resulting A.sub.301 when subtracted from the initial reading 
(after correction for dilution by added reagent) yields the 
hydroxylamine-extinguishable absorbance. Solutions of pure 
N-acetylthienamycin show a hydroxylamine-extinguishable absorbance of 
96.0%. 
ASSAY PROCEDURES FOR THIENAMYCIN 
I. Bioassay 
Assays of antibacterial activity are run according to the following 
disc-diffusion procedure unless otherwise indicated. The assay plates are 
prepared in the following manner. An overnight growth of the assay 
organism, Straphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% 
yeast extract is diluted with nutrient broth plus 0.2% yeast extract to a 
suspension having 55% transmittance at a wavelength of 660 m.mu.. This 
suspension is added to Difco nutrient agar supplemented with 2.0 g./l. 
Difco yeast extract, at 47.degree. C. to 48.degree. C., to make a 
composition containing 33.2 ml. of the suspension per liter of agar. Five 
ml. of this suspension is poured into petri dishes of 85 mm. diameter, and 
these plates are chilled and held at 4.degree. C. until used (5 day 
maximum). 
Samples of the antibiotic to be assayed are diluted to an appropriate 
concentration in phosphate buffer at pH 7. Filter-paper discs, 0.5-inch in 
diameter, are dipped into the test solution and placed on the surface of 
the assay plate; two discs for each sample are normally placed on one 
plate opposite to one another. The plates are incubated overnight at 
37.degree. C. and the zone of inhibition is measured as mm. diameter. The 
zone of inhibition measured in mm. determines relative potencies or, when 
compared with a purified reference standard such as cephalothin, the 
potency of antibiotic in units/ml. The unit of activity is based on 
cephalothin standard solutions of 8, 4, 2 and 1 .mu.g./ml. One unit is 
defined as the amount which calculates to produce the same inhibition as 1 
.mu.g. of cephalothin/ml. that zone of inhibition being between 16 and 21 
mm. diameter. 
II. Hydroxylamine-Extinguishable Absorbance 
The proportion of absorbance measured at 297 nm which can be attributed to 
the antibiotic content in impure samples is determined by the selective 
extinction of this absorbance (with concommitant inactivation of 
antibiotic activity) upon reaction with dilute hydroxylamine. 
Samples are prepared in 0.01 N potassium phosphate buffer at pH 7.0 to have 
an initial A.sub.297 between 0.05 and 2.0. Freshly prepared, neutral 
hydroxylamine (NH.sub.2 OH.HCl plus NaOH to a final pH of 7) is added to a 
final concentration of 10 mM., and reaction is allowed to progress at room 
temperature for at least 30 min. The resulting A.sub.297 when subtracted 
from the initial reading (after correction for dilution by added reagent) 
yields the hydroxylamine-extinguishable absorbance. Solutions of pure 
thienamycin show a hydroxylamine-extinguishable absorbance of 94.5%. 
ASSAY PROCEDURES FOR 890A.sub.1 and 890A.sub.3 
Bioassay 
An agar plate disc-diffusion method is employed using either Vibrio 
percolans ATCC 8461 or Salmonella gallinarum MB-1287 as tester organism. A 
purified sample of antibiotic 890A.sub.1 is used as standard. 
Plates containing Vibrio percolans ATCC 8641 are prepared as follows: 
A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml. 
of a sterilized medium containing 8 g./liter of Difco Nutrient Broth and 2 
g./liter of yeast extract in distilled water "nutrient broth-yeast 
extract" (herein after designated NBYE). The culture is incubated 
overnight on a rotary shaker at 28.degree. C. This culture is used to 
inoculate the surface of slants containing 1.5% agar in NBYE, and the 
inoculated slants are incubated overnight at 28.degree. C., and then 
stored in a refrigerator. 
The refrigerated slants prepared from a single lyophilized culture are used 
for up to four weeks from their preparation, as follows: A loop of 
inoculum from the slant is dispersed in 50 ml. of NBYE contained in a 250 
ml. Erlenmeyer flask. The culture is incubated overnight on a rotary 
shaker at 28.degree. C. and then diluted to a density giving 50% 
transmittance of 660 nm. A 33.2-ml. portion of this diluted culture is 
added to 1 liter of NBYE containing 15 g. of agar and maintained at 
46.degree. C. The inoculated agar-containing medium is poured into 
100.times.15 mm. plastic petri dishes, 5 ml. per dish, chilled, and 
maintained at 2.degree.-4.degree. C. for up to 5 days before using. 
Plates containing Salmonella gallinarum MB-1287 are prepared as follows: 
A sealed tube containing Salmonella gallinarum MB-1287 cells in skim milk, 
which had been frozen and lyophilized and sealed under vacuum, is opened 
and inoculated into 15 ml. of Brain Heart Infusion broth. The cells are 
allowed to grow without shaking at 37.degree. C. overnight, and the 
culture is inoculated onto slants of 0.8% BBL Nutrient broth+0.2% Difco 
yeast extract+1.5% agar. After growth overnight at 37.degree. C., a loop 
of the culture is transferred from the slant to a flask containing 50 ml. 
of 0.8% nutrient broth+0.2% yeast extract. The flask is shaken overnight, 
and 20 ml. of this culture is inoculated into one liter of 0.3% nutrient 
broth+0.2% yeast extract+1.5% agar which had been sterilized and cooled to 
48.degree. C. The inoculated NBYE agar is poured immediately into 
100.times.15 mm. plastic petri dishes, 5 ml. per dish, and the plates are 
kept at 0.degree.-3.degree. C. until use. 
Filter paper discs of one-half inch diameter are dipped into the solution 
to be assayed, and are placed on the agar. Alternatively, the discs may be 
loaded by pipetting one-tenth ml. of solution onto a dry disc, and then 
placing the disc on the agar. The diameter of the zone of inhibition is 
measured after appropriate incubation (9-18 hours at 37.degree. C. for 
Salmonella gallinarum MB-1287 or 12-24 hours at 25.degree. C. for Vibrio 
percolans ATCC 8461. If necessary, dilutions of the solutions to be 
assayed are made in 0.05 M potassium phosphate buffer, pH 7.4 "potassium 
phosphate buffer" (hereinafter referred to as KPB), or in deionized water. 
Calculations of potencies proceed as follows: a slope is determined by 
measuring the zone diameters of a solution of antibiotic 890A.sub.1 or 
890A.sub.3 and of a four-fold dilution (in KPB) of this solution. Two 
discs of each concentration are assayed on a single plate, and the average 
zone size at each concentration is determined. The slope is equal to 
one-half of the difference of the average zone sizes. Potencies are then 
calculated by the formula: Potency (units/ml.)= 
##EQU1## 
(Potency of Standard).times.Dilution.times.10 where D is the average 
diameter of the zones formed by the unknown, D.sub.s is the average 
diameter of the standard zones, and "Dilution" is the degree to which the 
unknown was diluted before assay. If no standard is used, D.sub.s is 
assumed to be 25 mm. and (Potency of Standard) is taken as 1 unit/ml., 
when measured on Vibrio percolans ATCC 8461. Pure 890A.sub.1 is defined as 
having a potency of 250 units per hydroxylamine-extinguishable absorbance 
unit at 300 nm, when used as a standard. Accordingly, the potency of 
antibiotic 890A.sub.3 is 31 units per hydroxylamine-extinguishable 
absorbance unit at 300 nm. 
For assays on Salmonella gallinarum MB-1287 plates, a slope is determined 
in the same way as on Vibrio percolans ATCC 8461 plates. When no standard 
is used, only relative potencies are calculated. If no slope is measured, 
a value of 2.3 mm. is assumed. The potency calculations proceed as with 
the Vibrio percolans ATCC 8461 assay. If no 890A.sub.1 standard is used, a 
control of penicillin G at 250 .mu.g/ml. may be employed in order to 
verify that the sensitivity of the organisms is in the normal range. 
II. Assay Procedure for Determining "890 Assay Units" 
The conventional disc-plate assay procedure is employed and the discs are 
1/2 inch in diameter. The routinely poured 10 ml./plate inoculated with 
Vibrio percolans (ATCC 8461) is used and the standard is cephaloridine. 
Four concentrations of cephaloridine constitute the standard--3.12, 6.25, 
12.5 and 25 mcg. per ml. with the 12.5 mcg. per ml. as a reference 
solution. The zone diameters on a 5 ml. plate for the standard are as 
follows: 
______________________________________ 
Conc. (mcg./ml.) 
Zone Diameter (mm.) 
______________________________________ 
3.12 16.8 
6.25 22.3 
12.5 25.0 
25 29.6 
______________________________________ 
A unit is defined as the amount of antibiotic-producing a 25 mm. zone of 
inhibition on a 5-ml. plate. Therefore, in this assay a concentration of 
12.5 mcgg per ml. of cephaloridine is considered equivalent to 1 unit of 
890A. Since the slope of the line for cephaloridine is 4.0, calculations 
of the potency of a sample are made using a slope of 4.0. 
III. Hydroxylamine Reaction 
Both antibiotics 890A.sub.1 and 890A.sub.3 react with hydroxylamine and 
produce a substance with greatly diminished absorbance at 300 nm. This 
provides the basis for a quantitative assay of the antibiotics 890A.sub.1 
and 890A.sub.3. 
The solution to be assayed is brought to 0.05 M in potassium phosphate, pH 
7.4 by adding 1/20th volume of a solution containing 0.8 M K.sub.2 
HPO.sub.4 and 0.2 M KH.sub.2 PO.sub.4. Then one-hundredth volume of 1 M 
hydroxylamine hydrochloride is added, and the absorbance at 300 nm is 
measured at intervals of one-half to two minutes. The reaction is 
conducted at 22.degree.-28.degree. C. First-order kinetics are assumed and 
a half-life is estimated from the absorbance decrease during the first ten 
minutes. From this half-life, the time is estimated beyond which no 
further absorbance decrease should be observed and observations are 
continued beyond that time. If no further decrease is observed beyond that 
time, the total absorbance decrease (correcting for dilution effect and 
absorbance of the hydroxylamine) is taken as the 
"Hydroxylamine-extinguishable absorbance at 300 nm (HAEA.sub.300)". If 
absorbance decrease is observed beyond that time, the rate of background 
absorbance decrease is calculated, and the observed decrease at that time 
is corrected for background decrease, assuming that background decrease is 
linear with time. The corrected value is then recorded as the 
HAEA.sub.300. 
The number of HAEA.sub.300 units is equal to the HAEA.sub.300 multiplied by 
the volume in ml.

EXAMPLE 1 
Method of Isolation of N-Acetylthienamycin amidohydrolase-producing 
Organisms 
A 1% (w/v) suspension of fertile lawn soil is prepared by suspending 1 gm. 
of lawn soil in 100 ml. sterile phosphate buffer-saline solution wherein 
the phosphate buffer-saline solution has the following composition: 
______________________________________ 
Phosphate Buffer-Saline Solution 
______________________________________ 
NaCl 8.8 g. 
1M Phosphate Buffer, pH 7.5* 
10 ml. 
Distilled H.sub.2 O 
______________________________________ 
*1M Phosphate Buffer, pH 7.5 
16 ml. 1M KH.sub.2 PO.sub.4 are mixed with 84 ml. 1M K.sub.2 HPO.sub.4. 
The pH of the phosphate buffer is adjusted to 7.5 by adding small 
quantities of either 1M KH.sub.2 PO.sub.4 or 1M K.sub.2 HPO.sub.4. 
Aliquots of this 1% stock soil suspension are used to prepare 10.times., 
100.times. and 1,000.times. dilutions. 
One-ml. portions of the stock suspension or 1-ml. portions of the 
10.times., 100.times. and 1,000.times. dilutions are added to 2-ml. 
portions of sterile, 1.0% agar solutions at 48.degree. C. The mixtures are 
quickly poured over the surface of sterile petri dishes of 85 mm. diameter 
containing 20 ml. of Medium A. Medium A has the following composition: 
______________________________________ 
Medium A 
______________________________________ 
KH.sub.2 PO.sub.4 3.0 g. 
K.sub.2 HPO.sub.4 7.0 g. 
MgSO.sub.4 0.1 g. 
Distilled H.sub.2 O 1000 ml. 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
For solid media: Add 20 g. agar The petri dishes are incubated for 18 days 
at 28.degree. C. Well-isolated colonies are picked and streaked on Medium 
B. Medium B has the following composition: 
______________________________________ 
Medium B 
______________________________________ 
Tomato paste 40 g. 
Ground oatmeal 15 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6 using NaOH 
______________________________________ 
For solid media: add 20 g. agar Individual clones are selected and grown 
for 2 days at 28.degree. C. on slants of Medium B. 
A portion of the growth of the slants is used to inoculate a 250 ml. 
Erlenmeyer flask containing 50 ml. of Medium A; a 250 ml. Erlenmeyer flask 
containing 50 ml. supplemented Medium B (supplemented after autoclaving 
with 0.4 m. of a membrane-sterilized solution of N-acetylethanolamine 
diluted 10.times. with water); and a 250 ml. Erlenmeyer flask containing 
50 ml. Medium C. Medium C has the following composition: 
______________________________________ 
Medium C 
______________________________________ 
Dextrose 20 g. 
Pharmamedia 8 g. 
Corn Steep Liquor 
(wet basis) 5 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7 with NaOH or HCl 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
The flasks are shaken at 23.degree. C. on a 220 rpm (2" throw) shaker for 4 
days. A 30-ml. portion from each flask is centrifuged for 15 minutes at 
8,000 rpm. The supernatant portion is removed, leaving only enough to form 
a thick suspension of cells and media solids. Half of the suspension is 
subjected to ultrasonic disruption using a Branson Instrument Model LS-75 
Sonifier with a 1/2 inch probe. The input power is set at position #4, and 
four successive 15 second cycles of irradition are used, while chilling 
the suspension in ice water during and between disruption. To test for the 
presence of N-acetylthienamycin amidohydrolase activity in either the 
whole cell preparation or the sonicate, aliquots of both whole cell 
suspensions and sonic disrupted suspensions are assayed by incubating 5 
.mu.l. portions of said suspensions with solutions containing 20 .mu.l of 
a 1.586 mg./ml. solution of N-acetylthienamycin in 10 mM potassium 
phosphate buffer, pH 7 and 10 .mu. l 0.2 M potassium phosphate buffer, pH 
7.4. Controls containing antibiotic and buffer alone and also cell 
suspensions without antibiotic are also run. After incubation of these 
mixtures overnight at 28.degree. C., 2 .mu.l aliquots are removed and 
applied on cellulose coated thin layer chromatography (TLC) plates, and 
the TLC plates developed in EtOH:H.sub.2 O, 70:30. After air drying, the 
TLC plates are placed on a Staphylococcus aureus ATCC 6538P assay plates 
for 5 minutes. The TLC plates are removed and the assay plates incubated 
overnight at 37.degree. C. 
The assay plates are prepared as follows: An overnight growth of the assay 
organism, Staphylococcus aereus ATCC 6538P, in nutrient broth plus 0.2% 
yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a 
suspension having 60% transmittance at a wavelength of 660 nm. This 
suspension is added to Difco nutrient agar supplemented with 2.0 g./l. 
Difco yeast extract at 47.degree. C. to 48.degree. C., to make a 
composition containing 33.2 ml. of the suspension per liter of agar. Forty 
ml. of this suspension is poured into 22.5 cm..times.22.5 cm. petri 
plates, and these plates are chilled and held at 4.degree. C. until used 
(5 day maximum). 
Activity of N-acetylthienamycin amidohydrolase in the incubation mixtures 
is indicated by the presence of a bioactive area of R.sub.f 0.44-0.47 due 
to thienamycin. The unreacted bioactive N-acetyl thienamycin appears at 
R.sub.f 0.7-0.89. The process of this Example provides one with the 
ability to isolate N-acetylethanolamine amidohydrolase producing 
microorganisms with N-acetylthienamycin amidohydrolase activity. 
EXAMPLE 2 
Deacetylation of N-Acetylthienamycin 
A 1% (w/v) suspension of fertile lawn soil is prepared by suspending 1 g. 
of lawn soil in 100 ml. sterile phosphate buffer-saline solution wherein 
the phosphate buffer-saline solution has the following composition: 
______________________________________ 
Phosphate Buffer-Saline Solution 
______________________________________ 
NaCl 8.8 g. 
1M Phosphate Buffer, pH 7.5* 
10 ml. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
*1M Phosphate Buffer, pH 7.5 
16 ml. 1M KH.sub.2 PO.sub.4 are mixed with 84 ml. 1M K.sub.2 HPO.sub.4. 
The pH of the phosphate buffer is adjusted to 7.5 by adding small 
quantities of either 1M KH.sub.2 PO.sub.4 or 1M K.sub.2 HPO.sub.4. 
Aliquots of this 1% stock soil suspension are used to prepare 10.times., 
100.times. and 1,000.times. dilutions. 
One-ml. portions of the stock suspension or 1-ml. portions of the 
10.times., 100.times. and 1,000.times. dilutions are added to 2-ml. 
portions of sterile, 1.0% agar solutions at 48.degree. C. The mixtures are 
quickly poured over the surface of sterile petri dishes of 85 mm. diameter 
containing 20 ml. of Medium A. Medium A has the following composition: 
______________________________________ 
Medium A 
______________________________________ 
KH.sub.2 PO.sub.4 3.0 g. 
K.sub.2 HPO.sub.4 7.0 g. 
MgSO.sub.4 0.1 g. 
Distilled H.sub.2 O 1000 ml. 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
For solid media: Add 20 g. agar The petri dishes are incubated for 18 days 
at 28.degree. C. A well-isolated colony is picked and streaked on a petri 
dish containing Medium B. Medium B has the following composition: 
______________________________________ 
Medium B 
______________________________________ 
Tomato paste 40 g. 
Ground oatmeal 15 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6 using NaOH 
______________________________________ 
For solid media: Add 20 g. agar An individual clone is selected and grown 
for 2 days at 28.degree. C. on a slant of Medium B. A portion of the 
growth on this slant is streaked on the surface of six slants prepared 
from Medium B. These slants are incubated for 2 days at 28.degree. C. This 
culture was identified as Protaminobacter ruber and has been designated 
MB-3528 in the culture collection of MERCK & CO., Inc., Rahway, New 
Jersey. 
A portion of the growth on the slant of Protaminobacter ruber MB-3528 is 
used to inoculate a 250-ml. Erlenmeyer flask containing 50 ml. of Medium 
C. Medium C has the following composition: 
______________________________________ 
Medium C 
______________________________________ 
Dextrose 20 g. 
Pharmamedia 8 g. 
Corn Steep Liquor 
(wet basis) 5 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7 with NaOH or HCl 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
The flask is shaken at 28.degree. C. on a 220 rpm (2" throw) shaker for 4 
days. A 25-ml. portion from the flask is centrifuged for 15 minutes at 
8,000 rpm. The supernatant is removed and the cells on the surface of the 
media solids scraped off into 0.5 ml. 0.05 M potassium phosphate buffer, 
pH 7.4. The resulting suspension is subjected to ultrasonic disruption 
using a Branson Instrument Model LS-75 Sonifier with a 1/2 inch probe at 
setting B 4 for 4, 15-second intervals, while chilling the suspension in 
ice water during and between disruption. A 10-.mu.l portion of the 
sonicate is mixed with a 25-.mu.l solution containing 840 .mu.g/ml. of 
N-acetylthienamycin in 10 mM potassium phosphate buffer, pH 7 and 
incubated overnight at 28.degree. C. Controls containing antibiotic and 
buffer alone; and sonicated cells and buffer without atibiotic are also 
run. After incubation overnight at 28.degree. C., 2-.mu.l quantities are 
applied on cellulose coated TLC plates, which are developed in 
EtOH:H.sub.2 O, 70:30. After air drying, the TLC plate is placed on a 
Straphyloccus aureus ATCC 6538P assay plate for 5 minutes. 
The assay plates are prepared as follows: An overnight growth of the assay 
organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% 
yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a 
suspension having 60% transmittance at a wavelength of 660 nm. This 
suspension is added to Difco nutrient agar supplemented with 2.0 g./l. 
Difco yeast extract at 47.degree. C. to 48.degree. C. to make a 
composition containing 33.2 ml. of the suspension per liter of agar. Forty 
ml. of this suspension is poured into 22.5 cm..times.22.5 cm. petri 
dishes, and these plates are chilled and held at 4.degree. C. until used 
(5 day maximum). 
The TLC plate is removed and the assay plate incubated overnight at 
37.degree. C. In addition to the unreacted bioactive N-acetylthienamycin 
spot at R.sub.f 0.70-0.89, a bioactive spot is observed at R.sub.f 
0.44-0.47 due to thienamycin. Control incubation mixtures of antibiotic 
plus buffer, cell sonicate plus buffer, and antibiotic plus buffer to 
which cell sonicate is added just prior to TLC application produce no 
bioactive material at R.sub.f 0.44-0.47. 
EXAMPLE 3 
Deacetylation of 890A.sub.1 
A portion of the growth on the slant of Protaminobacter ruber MB-3528 is 
used to inoculate a 250 ml. Erlenmeyer flask containing 50 ml. of Medium 
C. Medium C has the following composition: 
______________________________________ 
Medium C 
______________________________________ 
Dextrose 20 g. 
Pharmamedia 8 g. 
Corn Steep Liquor 
(wet basis) 5 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7 with NaOH or HCl 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
The flask is shaken at 28.degree. C. on a 220 rpm (2" throw) shaker for 4 
days. A 25-ml. portion from the flask is centrifuged for 15 minutes at 
8,000 rpm. The supernatant is removed and the cells on the surface of the 
media solids scraped off into 0.5 ml. 0.05 M potassium phosphate buffer, 
pH 7.4. The resulting suspension is subjected to ultrasonic disruption 
using a Branson Instrument Model LS-75 Sonifier with a 1/2 inch probe at 
setting 4 for 4, 15 second intervals, while chilling the suspension in ice 
water during and between disruption. A 10-.mu.l portion of the sonicate is 
mixed with 25 .mu.l of an 890A.sub.1 solution containing 4.85 
hydroxylamine-extinguishable optical density units at 300 nm/ml. Controls 
containing antibiotic and buffer alone; and sonicated cells and buffer 
without antibiotic are also run. After incubation overnight at 28.degree. 
C., 5 .mu.l quantities are applied on a cellulose coated TLC plate, which 
is developed in EtOH:H.sub.2 O, 70:30. After air drying, the TLC plate is 
placed on a Staphylococcus aureus ATCC 6538P assay plate for 5 minutes. 
The assay plates are prepared as follows: An overnight growth of the assay 
organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% 
yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a 
suspension having 60% transmittance at a wavelength of 660 nm. This 
suspension is added to Difco nutrient agar supplemented with 2.0 g./l. 
Difco yeast extract at 47.degree. C. to 48.degree. C., to make a 
composition containing 33.2 ml. of the suspension per liter of agar. Forty 
ml. of this suspension is poured into 22.5 cm..times.22.5 cm. petri 
plates, and these plates are chilled and held at 4.degree. C. until used 
(5 day maximum). 
The TLC plate is removed and the assay plate incubated overnight at 
37.degree. C. In addition to the unchanged bioactive 890A.sub.1 spot at 
R.sub.f 0.7-0.89, a new bioactive spot is observed at R.sub.f 0.44-0.47 
due to desacetyl 890A.sub.1. Control incubation mixtures of antibiotic 
plus buffer, and cell sonicate plus buffer produce no bioactive material 
at R.sub.f 0.44-0.47. 
EXAMPLE 4 
Deacetylation of 890A.sub.3 
Antibiotic 890A.sub.3 is deacetylated by the process described in Example 3 
for the deacetylation of 890A.sub.1 to provide desacetyl 890A.sub.3. 
EXAMPLE 5 
Preparation of desacetyl 890A.sub.1 
Six 250-ml. Erlenmeyer seed flasks containing 50 ml. each Medium C are 
inoculated with a portion of a slant of Protaminobacter ruber MB-3528. 
Medium C has the following composition: 
______________________________________ 
Medium C 
______________________________________ 
Dextrose 20 g. 
Pharmamedia 8 g. 
Corn Steep Liquor 
(wet basis) 5 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7 with NaOH or HCl 
N-Acetylethanolamine 
solution* 8.5 ml. 
______________________________________ 
*N-Acetylethanolamine Solution 
Nacetylethanolamine is diluted 10x in H.sub.2 O and membrane sterilized. 
This solution is added after autoclaving. 
The flasks are shaken at 28.degree. C. on a 220 rpm (2" throw) shaker for 1 
day. 
Forty-one 2-liter production flasks containing 400 ml. Medium C each are 
inoculated with 7 ml. per flask of the growth from these seed flasks. 
These production flasks are shaken at 28.degree. C. on a 220 rpm shaker 
(2" throw) for 6 days. The contents of the flasks are pooled and 
centrifuged at 8,000 rpm for 15 minutes. The cells are scraped off the 
media solids pellet into a final volume of 1,600 ml. 0.05 M potassium 
phosphate buffer, pH 7.4. This suspension is again centrifuged at 8,000 
rpm for 15 minutes. The cells are scraped off the media solids pellet into 
a final volume of 160 ml. 0.05 M potassium phosphate buffer, pH 7.4. This 
suspension is chilled to 5.degree. C. and aliquots of 15 ml. each are 
exposed to successive 15 second cycles of ultrasonic irradiation, 
employing the equipment described in Example 1, until no further 
diminution of turbidity is observed when a 500.times. dilution is made of 
the suspension into phosphate buffer-saline wherein the phosphate 
buffer-saline solution has the following composition: 
______________________________________ 
Phosphate Buffer-Saline Solution 
______________________________________ 
NaCl 8.8 g. 
1M Phosphate Buffer, pH 7.5* 
10 ml. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
*1M Phosphate Buffer, pH 7.5 
16 ml. 1M KH.sub.2 PO.sub.4 are mixed with 84 ml. 1M K.sub.2 HPO.sub.4. 
The pH of the phosphate buffer is adjusted to 7.5 by adding small 
quantities of either 1M KH.sub.2 PO.sub.4 or 1M K.sub.2 HPO.sub.4. 
To 1 liter of 0.005 M potassium phosphate buffer, pH 7.4, are added 250 mg. 
of the antibiotic 890A.sub.1. To this mixture is added 160 ml. of the 
sonic extract of Protaminobacter ruber containing N-acetylthienamycin 
amidohydrolase. The mixture is stirred slowly with a magnetic stirrer at 
28.degree. C. for 20 hours. The mixture is then centrifuged at 10,000 g. 
for 15 minutes and the supernatant removed, chilled to 5.degree. C., and 
adjusted to pH 4.5.+-.0.2 by the addition of acetic acid. Separation of 
desacetyl 890A.sub.1 from the unhydrolyzed antibiotic and from other 
constituents of the reaction mixture is effected in the following manner, 
a disc-diffusion bioassay against Staphylococcus aureus ATCC 6538P and 
measurements of hydroxylamine-extinguishable absorbance at 297 nm being 
used to monitor the performance of the purification procedures. (As 
described in Assay Procedures for Thienamycin). 
The acidified, centrifuged reaction mixture is absorbed at the rate of 12 
ml./min. on a 120 ml. bed of Dowex 50.times.4 sodium cycle, 20-50 mesh 
resin. The adsorbate is washed with 120 ml. of deionized water and then 
eluted with 2% aqueous pyridine at 6 ml./min. Following the emergence of 
75 ml. of the latter eluant from the column, the succeeding 240 ml. are 
pooled and concentrated to 25 ml. and the concentrate adjusted to pH 7. 
The 25 ml. concentrate is adsorbed at a rate of 1 ml./min. on a 25 ml. bed 
of Dowex-1.times.2, 50-100 mesh, chloride cycle resin. The resin is eluted 
with deionized water at the same rate. Following the emergence of 25 ml. 
of the eluant from the column, the succeeding 50 ml. are pooled, 
neutralized to pH 7 and concentrated to 10 ml. This concentrate is 
adjusted to pH 6.3 with acetic acid and is applied to a bed of Dowex 
50.times.8 (200-400 mesh) resin in the 2,6-lutidinium cycle, having a 
diameter of 1 cm. and height of 50 cm., which had previously been 
equilibrated with 0.1 M 2,6-lutidine acetate buffer, pH 6.3. Elution is 
conducted with the buffer at the rate of 1 ml./min. Following the 
emergence of 25 ml. of the eluant from the column, the succeeding 35 ml. 
are pooled and freeze-dried. 
The freeze-dried solids are dissolved in 0.5 ml. of 0.1 M 2,6-lutidine 
acetate buffer, pH 7.0. The solution is applied to a column of Bio-Gel P-2 
(200-400 mesh), having a diameter of 1 cm. and height of 50 cm., which had 
previously been equilibrated with this buffer. The gel is then developed 
with the same buffer at a rate of 0.5 ml./min. Following the emergence of 
25 ml. of eluant from the column, the succeeding 10 ml. are pooled and 
freeze-dried. 
The freeze-dried solids are dissolved in 4 ml. distilled water and applied 
on a 1.7 cm. diameter column packed with 90 ml. prewashed XAD-2 and 
equilibrated at 5.degree. C. with distilled water. The XAD-2 is washed 
prior to use successively with (1) 5 volumes of 1 N NaOH followed by 
deionized H.sub.2 O until effluent is neutral; (2) 5 volumes 1 N HCl 
followed by deionized H.sub.2 O until the effluent is neutral; (3) 5 
volumes each of methanol, acetone, 0.001 N EDTA tetrasodium, and finally, 
distilled H.sub.2 O. The sample is followed by two, 2-ml. portions of 
distilled water. The column is developed with distilled water at the rate 
of 2 ml./min. Four ml. fractions of eluate are collected. Fractions 25 
through 58 are pooled and lyophilized to yield desacetyl 890A.sub.1. 
EXAMPLE 6 
Preparation of N-Acetylthienamycin by Fermentation 
A tube of lyophilized culture of Streptomyces cattleya NRRL 8057 is opened 
aseptically and the contents suspended in 0.8 ml. of sterile Davis salts 
having the following composition: 
______________________________________ 
Davis Salts 
______________________________________ 
Sodium citrate 0.5 g. 
K.sub.2 HPO.sub.4 7.0 g. 
KH.sub.2 PO.sub.4 3.0 g. 
(NH.sub.4).sub.2 SO.sub.4 
1.0 g. 
MgSO.sub.4 . 7H.sub.2 O 
0.1 g. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
This suspension is used to inoculate four slants of Medium A (plus agar) 
having the following composition: 
______________________________________ 
Medium A 
______________________________________ 
Yeast Autolysate (Ardamine*) 
10.0 g. 
Glucose 10.0 g. 
Phosphate Buffer.sup.+ 2.0 ml. 
MgSO.sub.4 . 7H.sub.2 O 
0.05 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6.5 using NaOH 
______________________________________ 
*Ardamine: Yeast Products Corporation 
.sup.+ Phosphate Buffer Solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 95.0 g. 
Distilled H.sub.2 O 1000 ml. 
For Slants: add agar--25.0 g./l. The inoculated slants are incubated for 
one week at 28.degree. C. and then stored at 4.degree. C. 
Ten ml. of Medium A (without agar) is transferred aseptically to one of 
these slants, the spores and aerial mycelia scraped into suspension, and 
1.2 ml. of this suspension used to inoculate three 2-liter baffled 
Erlenmeyer flasks containing 500 ml. of Medium A (without agar). These 
seed flasks are shaken at 28.degree. C. on a 160 rpm shaker for 24 hours 
at which time the growth is satisfactory. 
The growth from these seed flasks is pooled and used to inoculate a 
756-liter stainless steel fermentor containing 467 liters of Medium A 
(without agar). This tank is operated at 28.degree. C. using an agitation 
rate of 130 rpm and an airflow of 10 cu. ft. per minute for 24 hours. 
Defoamer, Polyglycol 2000 (Dow Chemical Corp.), is used as required but 
does not exceed 0.1%. pH determinations are made as follows: 
______________________________________ 
Age, Hours 0 24 
______________________________________ 
pH 6.3 6.4 
______________________________________ 
Four hundred fifty-four liters of the growth in this seed tank is used to 
inoculate a 5,670-liter stainless steel fermentor containing 4,082 liters 
of Medium E, wherein Medium E has the composition: 
______________________________________ 
Medium E 
______________________________________ 
Cerelose 25.0 g. 
Corn Steep Liquor (wet basis) 
15.0 g. 
Distiller's Solubles 10.0 g. 
Cottonseed Media (Pharmamedia) 
5.0 g. 
CoCl.sub.2 . 6H.sub.2 O 0.01 g. 
CaCO.sub.3 (after pH adjustment) 
3.0 g. 
Polyglycol 2000 0.25% 
Tap water 1000 ml. 
pH adjusted to 7.3 using NaOH 
______________________________________ 
This tank is run at 24.degree. C. using an agitation rate of 70 rpm and an 
airflow of 54.3 cu. ft. per minute for 138 hours. Additional defoamer, 
Polyglycol 2000, is added as required, but does not exceed 0.1%. 
Antibacterial assays are run and the data is as follows: 
______________________________________ 
ATCC No. 6633 
(3/8" disc.) 
Age pH (mm.) 
______________________________________ 
0 6.9 0 
24 6.3 0 
36 6.0 0 
48 5.9 0 
60 6.0 23 
72 5.9 -- 
84 6.0 21 
96 6.2 -- 
108 6.5 35 
120 6.6 36 
132 6.7 41 
138 6.7 39 
______________________________________ 
The 4082 liters of fermentation broth is filtered using a 30-inch filter 
press and a filter aid admix to the extent of 4% w/v. A 46 g. amount of 
ethylenedinitrile tetraacetic acid (EDTA), sodium salt is added to the 
filtrate. The filtrate is cooled to 6.degree. C., adjusted to pH 
4.5.+-.0.2 and maintained at 6.degree. C. The cold filtrate is applied to 
a 480 liter column of Dowex 50.times.4 Na.sup.+, 20-50 mesh at about 48 
l./minute. After a 1400 liter forerun has passed through, 18.9 liters of 
spent is collected, the pH of the spent is adjusted to 7.08 with NaOH, and 
stored at 5.degree. C. 
A 3.8-cm. diameter column packed with 300 ml. of Dowex 1.times.2, 50-100 
mesh, resin in the chloride cycle is prepared and washed with 600 ml. of 
deionized water at 5.degree. C. Four liters of the cold Dowex 50.times.4 
spent is passed through the column at the rate of 30 ml./minute. The 
column is washed with 300 ml. 25 .mu.M EDTA. The antibiotic, 
N-acetylthienamycin, is eluted at 5.degree. C. at the rate of 15 
ml./minute with 900 ml. 5% NaCl solution containing 0.01 M potassium 
phosphate buffer, pH 7.0, and 25 .mu.M EDTA. Fourteen fractions of 75 ml. 
are collected and assayed for biological activity by the disc-diffusion 
procedure. Eluate fractions 3 through 9, comprising 525 ml. are pooled and 
concentrated under vacuum to 115 ml. The concentrate contains 65% of the 
total bioactive material applied on the Dowex 1.times.2 Cl.sup.- column. 
The Dowex 1.times.2 Cl.sup.- eluate concentrate is applied at 5.degree. C. 
on a 3.8 cm. diameter column packed with 450 ml. prewashed XAD-2. The 
XAD-2 is prewashed columnwise successively with 4 column volumes: 
(1) 0.001 M EDTA, (2) 1 N NaOH, (3) deionized H.sub.2 O, (4) 1 N HCl, (5) 
deionized H.sub.2 O, (6) methanol, (7) acetone, (8) deionized water and 
prior to use with 2250 ml. 5% NaCl solution containing 25 .mu.M EDTA. 
After the sample is applied to the column, it is followed by two 25-ml. 
portions of H.sub.2 O. The column is developed at 5.degree. C. with 
deionized H.sub.2 O at a flow rate of 10 ml./minute. The first fraction 
contains 400 ml. and 11 additional fractions of 75 ml. are collected. The 
pH of each fraction is adjusted to between 6.9 and 7.13 with 1 N NaOH or 1 
N HCl Fractions 3 to 9, containing 46% of the total bioactive material 
applied to the XAD-2 column, are combined and have a total volume of 490 
ml. A sample of 45 ml. is removed for bioassays by the standard 
disc-diffusion procedure against Staphylococcus aureus ATCC 6538P. The 
remaining 445 ml. is concentrated under vacuum to 50 ml. 
The 50 ml. XAD-2 eluate concentrate is pumped at 5.degree. C. onto a 1.5 
cm. column packed with 40 ml. prewashed Dowex 1.times.4 Cl.sup.-, minus 
400, at 5.degree. C. and at a rate of 1 ml./min. The Dowex 1.times.4 
Cl.sup.-, minus 400 (defined by decanting from water) resin is washed 
column-wise prior to use with 240 ml. 0.2 M NaCl containing 0.005 M 
NH.sub.4 Cl and 0.1 mM NH.sub.4 OH at the rate of 1 ml./minute and then 
with 120 ml. deionized water at the same rate. 
After the sample is applied to the column, it is followed by two, 5-ml. 
portions of deionized water. The column is developed at 5.degree. C. at 
the rate of 0.92 ml./min. with 0.07 M NaCl containing 0.005 M NH.sub.4 Cl 
and 0.1 mM NH.sub.4 OH. Fractions of 8.6 to 9.3 ml. are collected. 
Fractions obtained after 600 ml. of eluate have been collected and ending 
with 710 ml. are pooled and contain 98% of the total bioactive material 
applied on the Dowex 1.times.4 column. This pool is concentrated under 
vacuum to 2 ml. 
The Dowex 1.times.4 concentrate is applied to a 2.2 cm. diameter column 
packed with 225 ml. Bio-Gel P-2, 200-400 mesh, with an exclusion limit of 
1800 Daltons (defined prior to use by decantation from distilled water). 
The Bio-Gel P-2 column is washed prior to use with 225 ml. 1 M NaCl 
followed by 100 ml. deionized water. The column is developed with 
5.degree. C. deionized water at the rate of 1 ml./minute, and two ml. 
fractions are collected. Fractions from 104 ml. to 128 ml. eluate, 
containing 83% of the bioactivity applied to the Bio-Gel P-2 column, are 
combined and concentrated to 1.58 ml. 
A 50 ml. XAD-2 column (1.6 cm..times.27 cm.) is prepared and prewashed 
column-wise with 200 ml. of 1 mM EDTA, 1 N NaOH, deionized water, 1 N HCl, 
deionized water, methanol, acetone, and deionized water. The Bio-Gel P-2 
concentrate containing 44.4 hydroxylamine-extinguishable optical density 
units is applied to the XAD-2 column at 5.degree. C. and is followed by 
two, 2-ml. portions of deionized water. The column is washed with 
deionized water at the rate of 1 ml./minute until the UV absorbance at 300 
nm of the washings is reduced to 0.060. The column is eluted with 50% 
methanol in deionized water at the rate of 1 ml./minute and 1-ml. 
fractions are collected. Fractions having absorbance at 300 nm over 0.1 
are combined and concentrated under vacuum to give the product, 
N-acetylthienamycin containing 11.6 hydroxylamine-extinguishable O.D. 
units. 
EXAMPLE 7 
Preparation of N-Acetylthienamycin by Fermentation 
A tube of lyophilized culture of Streptomyces cattleya MA-4297 is opened 
aseptically and the contents suspended in 0.8 ml. of sterile Davis salts 
having the following composition: 
______________________________________ 
Davis Salts 
______________________________________ 
Sodium citrate 0.5 g. 
K.sub.2 HPO.sub.4 7.0 g. 
KH.sub.2 PO.sub.4 3.0 g. 
(NH.sub.4).sub.2 SO.sub.4 
1.0 g. 
MgSO.sub.4 . 7H.sub.2 O 
0.1 g. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
This suspension is used to inoculate four slants of Medium A (plus agar) 
having the following composition: 
______________________________________ 
Medium A 
______________________________________ 
Yeast Autolysate (Ardamine*) 
10.0 g. 
Glucose 10.0 g. 
Phosphate Buffer.sup.+ 2.0 ml. 
MgSO.sub.4 . 7H.sub.2 O 
0.05 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6.5 using NaOH 
______________________________________ 
*Ardamine: Yeast Products Corporation 
.sup.+ Phosphate Buffer Solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 95.0 g. 
Distilled H.sub.2 O 1000 ml. 
For Slants: add agar--25.0 g./l. 
The inoculated slants are incubated for one week at 28.degree. C. and then 
stored at 4.degree. C. 
Ten ml. of Medium A is transferred aseptically to one of these slants, the 
spores and aerial mycelia scraped into suspension, and 1.2 ml. of this 
suspension used to inoculate three 2-liter baffled Erlenmeyer flasks 
containing 500 ml. of Medium A (without agar). These seed flasks are 
shaken at 28.degree. C. on a 160 rpm shaker for 24 hours at which time the 
growth is satisfactory. 
The growth from these seed flasks is pooled and used to inoculate a 756 
liter stainless steel fermentor containing 467 liters of Medium A (without 
agar). This tank is operated at 28.degree. C. using an agitation rate of 
130 rpm and an airflow of 10 cu. ft. per minute for 24 hours. Defoamer, 
Polyglycol 2000 (Dow Chemical Corp.), is used as required but does not 
exceed 0.1%. pH determinations are made as follows: 
______________________________________ 
Age, Hours 0 24 
______________________________________ 
pH 6.3 6.4 
______________________________________ 
Four hundred fifty-four liters of the growth in this seed tank is used to 
inoculate a 5,670 liter stainless steel fermentor containing 4,082 liters 
of Medium E, wherein Medium E has the composition: 
______________________________________ 
Medium 
Medium E 
______________________________________ 
Cerelose 25.0 g. 
Corn Steep Liquor (wet basis) 
15.0 g. 
Distiller's Solubles 10.0 g. 
Cottonseed Media (Pharmamedia) 
5.0 g. 
CoCl.sub.2 . 6H.sub.2 O 0.01 g. 
CaCO.sub.3 (after pH adjustment) 
3.0 g. 
Polyglycol 2000 0.25% 
Tap water 1000 ml. 
pH adjusted to 7.3 using NaOH 
______________________________________ 
This tank is run at 24.degree. C. using an agitation rate of 70 rpm and an 
airflow of 54.3 cu. ft. per minute for 138 hours. Additional defoamer, 
Polyglycol 2000, is added as required, but does not exceed 0.1%. 
Antibacterial assays are run and the data is as follows: 
______________________________________ 
ATCC No. 6633 
(3/8" disc.) 
Age pH (mm.) 
______________________________________ 
0 6.9 0 
24 6.3 0 
36 6.0 0 
48 5.9 0 
60 6.0 23 
72 5.9 -- 
84 6.0 21 
96 6.2 -- 
108 6.5 35 
120 6.6 36 
132 6.7 41 
138 6.7 39 
______________________________________ 
The 4082 liters of fermentation broth is filtered using a 30-inch filter 
press and a filter aid admix to the extent of 4% w/v. A 46 g. amount of 
ethylenedinitrile tetraacetic acid (EDTA), sodium salt is added to the 
filtrate. The filtrate is cooled to 6.degree. C., adjusted to pH 
4.5.+-.0.2 and maintained at 6.degree. C. The cold filtrate is applied to 
a 480 liter column of Dowex 50.times.4 Na.sup.+, 20-50 mesh at about 48 
l./minute. After a 1400 liter forerun has passed through, 18.9 liters of 
spent is collected, the pH of the spent is adjusted to 7.08 with NaOH, and 
stored at 5.degree. C. 
A 3.8 cm. diameter column packed with 300 ml. of Dowex 1.times.2, 50-100 
mesh, resin in the chloride cycle is prepared and washed with 300 ml. of 
deionized water at 5.degree. C. Four liters of the cold Dowex 50.times.4 
spent is passed through the column at the rate of 30 ml./minute. The 
column is washed with 350 ml. 25 .mu.M EDTA and then eluted at 5.degree. 
C. with 900 ml., 5% NaCl solution containing 0.01 N Tris.HCl, pH 7, and 25 
.mu.M EDTA at the rate of 15 ml./minute. Fractions of 75 ml. are collected 
and assayed by the disc-diffusion procedure against Staphylococcus aureus 
ATCC 6538P. Fractions 4 to 10 containing 47% of the bioactivity applied 
are added to 42.5 ml. of the sample removed for bioassay from the first 
XAD-2 pool described in Example 6. These combined fractions are 
concentrated under vacuum to 100 ml., and the pH adjusted to 6.32 with 
HCl. 
A 3.8-cm. diameter column packed with 450 ml. XAD-2 resin is prewashed 
column-wise successively with 4 column volumes: (1) 0.001 M EDTA, (2) 1 N 
NaOH, (3) deionized H.sub.2 O, (4) 1 N HCl, (5) deionized H.sub.2 O, (6) 
methanol, (7) acetone, (8) deionized water and washed prior to use with 
2250 ml. 5% NaCl solution containing 25 .mu.M EDTA. The above concentrate 
is applied to the XAD-2 column and is followed by two, 5-ml. portions of 
deionized water. The column is developed at 5.degree. C. at the rate of 10 
ml./minute with deionized water. The first fraction contains 40 ml., and 
subsequent fractions of 75 ml. are collected and assayed by the 
disc-diffusion process. Fractions 9 to 15, containing 22% of the 
bioactivity applied on the XAD-2 column are pooled and concentrated under 
vacuum to 56 ml. 
A 21 cm..times.1.7 cm. column packed with 40 ml. of Dowex 1.times.4 
Cl.sup.-, minus 400 mesh, (defined by decanting from water) is washed 
column-wise prior to use with 240 ml. 0.2 M NaCl containing 0.005 M 
NH.sub.4 Cl and 0.1 mM NH.sub.4 OH at the rate of 1 ml./minute and then 
with 120 ml. deionized water at the same rate. 
The XAD-2 concentrate is applied on the column and is followed by two, 
2-ml. portions of deionized water and then by two, 2-ml. portions of 
eluting buffer. The column is eluted at 5.degree. C. with a solution 0.07 
M NaCl containing 0.005 M NH.sub.4 Cl and 0.1 mM NH.sub.4 OH at the rate 
of 1 ml./minute. Fractions of 10 ml. are collected and assayed by the disc 
diffusion method. Eluate fractions from 544 ml. through 647 ml. containing 
an apparent 100% of the applied bioactivity are combined and concentrated 
under vacuum to 2.3 ml. The concentrate contains 36.4 
hydroxylamine-extinguishable optical density units. 
A 2.2 cm..times.62 cm. column packed with 225 ml. Bio-Gel P-2, 200-400 mesh 
resin with an exclusion limit of 1800 Daltons is washed prior to use with 
225 ml. 1 M NaCl followed by 100 ml. deionized water. The Dowex 1.times.4 
concentrate is applied to the column and is followed by two, 2-ml. 
portions of deionized water. The column is developed at 5.degree. C. with 
deionized water at the rate of 1 ml./minute and 2 ml. fractions are 
collected and assayed by the disc-diffusion procedure. Fractions from 124 
ml. to 129 ml., containing 7.04 hydroxylamine-extinguishable O.D. units 
are combined and concentrated under vacuum to 2 ml. to give an aqueous 
solution of the product, N-acetylthienamycin. Fractions from 117 to 123 
ml. and 130 to 139 ml. are combined to give a solution of the product, 
N-acetylthienamycin containing 13.7 hydroxylamine-extinguishable O.D. 
units. Deacetylation of this material obtained herewith in accordance with 
the process of Example 2 affords the antibiotic thienamycin. 
EXAMPLE 8 
Preparation of Thienamycin 
A tube of lyophilized culture of Streptomyces cattleya MA-4297 is opened 
aseptically and the contents suspended in 50 ml. of sterile Medium A 
contained in a 250-ml. baffled Erlenmeyer flask. Medium A has the 
following composition: 
______________________________________ 
Medium A 
______________________________________ 
Yeast Autolysate (Ardamine*) 
10.0 g. 
Glucose 10.0 g. 
Phosphate Buffer.sup.+ 2.0 ml. 
MgSO.sub.4 . 7H.sub.2 O 
0.05 g. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6.5 using NaOH 
______________________________________ 
*Ardamine: Yeast Products Corporation 
.sup.+ Phosphate Buffer Solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 95.0 g. 
Distilled H.sub.2 O 1000 ml. 
The inoculated flask is shaken at 28.degree. C. on a 220 rpm (2" throw) for 
48 hours. Forty ml. of the 48-hour broth is removed aseptically and mixed 
with 40 ml. of sterile 20% (v/v) aqueous glycerol. Two-ml. quantities of 
the resulting mixture are pipetted into sterile 1-gram vials which are 
then frozen and stored in the vapor phase of a liquid nitrogen freezer. 
Frozen vial contents are used to inoculate a 250 ml. baffled Erlenmeyer 
flask containing 50 ml. of Medium A. This seed flask is shaken at 
28.degree. C. on a 160 rpm shaker at 28.degree. C. for 24 hours. 
Ten-ml. portions from this seed flask are used to inoculate 2-liter baffled 
Erlenmeyer flasks containing 500 ml. of Medium A. These seed flasks are 
shaken on a 160 rpm shaker at 28.degree. C. for 24 hours. 
A one thousand-ml. portion of the pooled contents of these seed flasks is 
used to inoculate a 756-liter stainless steel fermentor containing 467 
liters of Medium A. This tank is operated at 28.degree. C. using an 
agitation rate of 130 rpm and an airflow of 10 cu. ft. per minute for 24 
hours. Polyglycol 2000 (Dow Chemical Corp.) is used as required as a 
defoamer but not to exceed 0.1%. Measurements of pH and dextrose are made 
and are as follows: 
______________________________________ 
Age: Hours 0 12 24 
______________________________________ 
pH 6.4 6.4 6.6 
Dextrose mg./ml. 
8.1 8.1 8.1 
______________________________________ 
Four hundred fifty-three liters of this growth are used to inoculate a 
5670-liter stainless steel fermentor containing 4082 liters of Medium E, 
wherein Medium E has the composition: 
______________________________________ 
Medium E 
______________________________________ 
Cerelose 25.0 g. 
Corn Steep Liquor (wet basis) 
15.0 g. 
Distiller's Solubles 10.0 g. 
Cottonseed Media (Pharmamedia) 
5.0 g. 
CoCl.sub.2 . 6H.sub.2 O 0.01 g. 
CaCO.sub.3 (after pH adjustment) 
3.0 g. 
Polyglycol 2000 0.25% 
Tap water 1000 ml. 
pH adjusted to 7.3 using NaOH 
______________________________________ 
This tank is operated at 24.degree. C. using an agitation rate of 70 rpm 
and an airflow of 54.3 cu. ft. per minute for 144 hours. Defoamer, 
Polyglycol 2000, is added as required but does not exceed 0.1%. 
Centrifuged broth is assayed against Staphylococcus aureus ATCC 6538P by 
the standard disc-diffusion procedure. The results are tabulated in the 
table below under the heading "Antibiotic Activity vs ATCC 6538P". Assays 
are also run by the disc-diffusion procedure using 3/8-inch filter-paper 
discs and 10 ml. assay plates and the results tabulated in the table below 
under the heading "Antibiotic Activity (10 ml. plates)". 
The 10 ml. assay plates are prepared as follows: An overnight growth of the 
assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 
0.2% yeast extract is diluted with nutrient broth plus 0.2% yeast extract 
to a suspension having 40% transmittance at a wavelength of 660 m.mu.. 
This suspension is added to Difco nutrient agar supplemented with 2.0 
g./l. Difco yeast extract, at 47.degree. C. to 48.degree. C., to make a 
composition containing 33.2 ml. of the suspension per liter of agar. Ten 
ml. of this suspension are poured into petri plates of 85 mm. diameter, 
and the plates are chilled and held at 4.degree. C. until used (5 day 
maximum). 
______________________________________ 
Antibiotic 
Antibiotic 
Activity vs 
Activity 
ATCC 6538P 
(10 ml. plates) 
Age pH Dextrose mg./ml. 
(mm.) (mm.) 
______________________________________ 
0 6.6 22.2 
12 6.3 20.2 
24 5.8 18.0 0 
36 6.0 13.2 21.5 
48 6.0 8.6 21.5 
60 5.7 6.4 26.5 
72 5.8 2.7 25.5 
84 6.2 0.3 27.5 
96 6.4 0.2 36.0 
108 6.4 0 35.0 
120 6.3 41.5 37.0 
132 5.8 37.5 
144 5.9 43.0 37.5 
______________________________________ 
The 4082 liters of fermentation broth is filtered using a 30-inch filter 
press and a filter aid admix to the extent of 4% w/v. A 12 g. amount of 
(ethylenelinitrilo) tetraacetic acid, sodium salt is added to the 
filtrate. The filtrate is cooled to 6.degree. C., adjusted to pH 
4.5.+-.0.2 and maintained at 6.degree. C. The cold filtrate is adsorbed on 
480 l. of Dowex 50.times.4 Na.sup.+, 20-50 mesh at about 48 l./min. The 
adsorbate is washed with 480 l. of deionized water and then eluted with 2% 
aqueous pyridine at 24 l./min. and three fractions of 300 l., 520 l. and 
240 l. are collected and assayed at pH 7.0. The assays indicate that the 
eluate fractions contain 4%, 16% and 6%, respectively of the bioactivity 
applied on the Dowex 50.times.4 Na.sup.+ column. Eluate fraction two is 
concentrated to 48 l. and adjusted to pH 7. 
The 48 l. concentrate is adjusted to pH 7.3 and adsorbed on 76 l. of Dowex 
1.times.2, 50 to 100 mesh, chloride cycle resin at 7.6 l./min. The resin 
is eluted with deionized water at the same rate. Four fractions are 
collected, two of 48 l., one of 70 l. and one of 48 l. The fractions are 
adjusted to pH 7 as collected. Assays indicate that 68% of the starting 
bioactivity is in the 70 l. fraction. This fraction is concentrated to 18 
l. at pH 7.0 and filtered using a 0.45 micron Millipore Filter. The 
filtrate is tray freeze-dried to yield 99 grams of product having a 
potency of 310 units/mg. 
Ten g. of the freeze-dried solids are taken up in 0.1 M 2,6-lutidine 
acetate buffer, pH 6.3. The solution, 125 ml. readjusted to pH 6.3 with 
acetic acid, is applied to a column of Dowex 50.times.8 (200-400 mesh) in 
the 2,6-lutidine cycle, 7.6.times.142 cm., which had previously been 
equilibrated with buffer, and developed with 0.1 M buffer at 25 ml./min. A 
3-1. fore-cut is collected followed by 200 fractions of 20 ml. each. Every 
fourth fraction 36 through 192 is assayed at a dilution of 1:200. The 
bioactivity is contained in fractions 56 through 192, reaching a maximum 
in fractions 92 through 96. Fractions 80 through 136 are combined and 590 
ml. of deionized water added to give 1760 ml. The pooled, diluted, 
solution containing 62% of the starting bioactivity applied on the Dowex 
50.times.8 column, is freeze-dried. 
The freeze-dried solids are dissolved in 0.1 M 2,6-lutidine acetate, pH 7.0 
buffer. The solution, 27 ml., is applied to a column of Bio-Gel P-2 
(200-400 mesh) 5.times.112 cm. which had previously been equilibrated with 
0.1 M buffer. The gel is then developed with the same buffer at 10 
ml./min. 
The effluent stream is monitored with a Meccomatic recording differential 
refractometer. The development is continued until 105 fractions, 20 ml. 
each, are collected. Every fraction, 70 through 93, is assayed at a 
dilution of 1:300. The bio-activity is found in fractions 73 through 82, 
reaching a maximum in fractions 77 and 78. Fractions 75 through 80 are 
freeze-dried to obtain 90 mg. of antibiotic with an average potency of 
10,000 units/mg. 
The 90 mg. of freeze-dried solid is taken up into 4 ml. of 0.01 M potassium 
phosphate buffer, pH 7. This solution, containing 596 
hydroxylamine-extinguishable optical density units (this measure of the 
thienamycin content being described in Section II of Assay Procedures for 
Thienamycin) is applied on a 1.7-cm. diameter column packed with 90 ml. 
prewashed XAD-2 and equilibrated prior to use with 180 ml. of 0.01 M 
potassium phosphate buffer, pH 7, at 5.degree. C. The XAD-2 is washed 
prior to use successively with (1) 5 volumes of 1 N NaOH followed by 
deionized H.sub.2 O until effluent is neutral; (2) 5 volumes 1 N, 
--followed by deionized H.sub.2 O until the effluent is neutral; (3) 5 
volumes each of methanol, acetone, 0.001 M EDTA tetrasodium, and finally 
distilled H.sub.2 O. Vacuum is applied to all solvents before use. 
After the sample is applied on the column it is followed by two, 2-ml. 
portions of the phosphate buffer. The column is developed at 5.degree. C. 
with the buffer at a flow rate of 2 ml./min. Four-ml. fractions of eluate 
are collected. Fractions obtained after 100 ml. of eluate has been 
collected and ending with 253 ml. are combined and concentrated on a 
rotary evaporator under vacuum and below 10.degree. C. to a volume of 6 
ml. 
This solution, containing 436 hydroxylamine-extinguishable optical density 
units, is applied on a 1.7 cm. diameter column packed with 90 ml. XAD-2 
prewashed as above and equilibrated at 5.degree. C. with distilled water. 
The sample is followed by two, 2-ml. portions of distilled water. The 
column is developed with distilled water at the rate of 2 ml./min. 
Four-ml. fractions of eluate are collected. Fractions obtained after 100 
ml. of eluate has been collected and ending with 151 ml. are pooled and 
concentrated on a rotary evaporator to a volume of 2.73 ml. and the 
solution lyophilized to yield 6.49 mg. of thienamycin. Fractions obtained 
between 152 ml. and 345 ml. are pooled and concentrated on a rotary 
evaporator to a volume of 3.34 ml. and lyophilized to yield 11.53 mg. of 
thienamycin. These fractions contain a total of 369 
hydroxylamine-extinguishable optical density units. This represents a 3.1 
fold purification over the material applied to the first XAD-2 column and 
yields a calculated potency of 31,000 units/mg. Spectrophotometric 
analysis of a sample of this product shows an E.sub.1 cm..sup.1% =253 when 
measured in phosphate buffer, pH 7, at 297 nm. 
A ten-g. portion of the 99 g. freeze-dried solids obtained by the Dowex 
1.times.2 purification above is taken up in 0.1 M 2,6-lutidine acetate 
buffer, pH 6.3. The solution, 125 ml., readjusted to pH 6.3 with acetic 
acid, is applied to a 7.6.times.142 cm. column of Dowex 50.times.8 in the 
2,6-lutidine cycle, which had previously been equilibrated with buffer. 
The column is developed with 0.1 M buffer at 35 ml./min. A 3.6-1. fore-cut 
is collected followed by 200 fractions of 20 ml. each. Every fourth 
fraction 6 through 194 is assayed at a dilution of 1:200. The bioactivity 
is contained in fractions 18 through 178, reaching a maximum in fractions 
62 through 82. Fractions 42 through 102 are combined and 640 ml. of 
deionized water added to give 1920 ml. The pooled, diluted, solution 
containing 63% of the bio-activity applied on the Dowex 50.times.8 column, 
is freeze-dried. 
The freeze-dried solids are dissolved in 0.1 M 2,6-lutidine acetate, pH 7.0 
buffer. The solution, 25 ml., is applied to a 5.times.112 cm. column of 
Bio-Gel P-2 (200-400 mesh), which had previously been equilibrated with 
0.1 M buffer. The gel is then developed with the same buffer at 10 
ml./min. 
The effluent stream is monitored with a Meccomatic recording differential 
refractometer. The development is continued until 125 fractions, 20 ml. 
each, are collected. Every fraction, 70 through 89, is assayed at a 
dilution of 1:300. The bio-activity is found in fractions 72 through 81, 
reaching a maximum in fraction 77. Fractions 75 through 79 are 
freeze-dried to obtain 100.5 mg. of antibiotic with a potency of 8320 
units/mg. 
The 100.5 mg. of freeze-dried solid is taken up into 4 ml. of 0.01 M 
potassium phosphate buffer, pH 7. This solution, containing 692 
hydroxylamine-extinguishable optical units is applied on a 1.7-cm. 
diameter column packed with 90 ml. prewashed XAD-2 and equilibrated prior 
to use with 180 ml. of 0.01 M potassium phosphate buffer, pH 7, at 
5.degree. C. The XAD-2 is washed prior to use successively with (1) 5 
volumes of 1 N NaOH followed by deionized H.sub.2 O until effluent is 
neutral; (2) 5 volumes 1 N HCl followed by deionized H.sub.2 O until the 
effluent is neutral; (3) 5 volumes each of methanol, acetone, 0.001 M EDTA 
tetrasodium, and finally distilled H.sub.2 O. Vacuum is applied to all 
solvents before use. 
After the sample is applied on the column it is followed by two 2-ml. 
portions of the phosphate buffer. The column is developed at 5.degree. C. 
with the buffer at a flow rate of 2 ml./min. Four-ml. fractions of eluate 
are collected. Fractions obtained after 109 ml. of eluate has been 
collected and ending with the 309th ml. are combined. To this combined 
eluate is added the 11.53-mg. sample of XAD-2 purified antibiotic obtained 
above comprising 186 hydroxylamine-extinguishable optical density units. 
The combined eluate together with the added antibiotic is concentrated in 
vacuo on a rotary evaporator at a temperature below 10.degree. C. to a 
volume of 7 ml. 
This solution, containing 720 hydroxylamine-extinguishable optical density 
units is applied on a 1.7-cm. diameter column packed with 90 ml. XAD-2 
prewashed as above and equilibrated at 5.degree. C. prior to use with 
distilled water. The sample is followed by two, 2-ml. portions of 
distilled water. The column is developed with distilled water at the rate 
of 2 ml./min. Four-ml. fractions of eluate are collected. Fractions 
obtained after 109 ml. of eluate have been collected and ending with the 
301st ml. are pooled and concentrated on a rotary evaporator to a volume 
of 10.3 ml. This solution, containing 589 hydroxylamine-extinguishable 
optical density units, is lyophilized to yield 23.6 mg. of antibiotic with 
a calculated potency of 30,140 units/mg. 
The antibiotic thienamycin thus prepared is a white, amorphous solid with a 
fibrous consistency, a sample of which on exposure in a glass capillary 
tube to temperatures elevated at a rate of 3.degree. C. per minute, 
underwent decomposition without the intervention of a liquid phase in the 
following stages: softening occurred at 130.degree. to 140.degree. C. with 
a contraction in volume of the solid continuing until 170.degree. to 
174.degree. C. in which range the material yellowed; sintering and a 
progressive intensification of color to reddish-brown being observed in 
the range 180.degree. to 200.degree. C. and finally carbonization and 
residual traces of solid being found at 205.degree. C. 
A further sample of this material on spectrophotometric analysis shows an 
absorbance peak at 296.5 nm with an E.sub.1 cm..sup.1% =268.2. Elemental 
analysis yields the following results: (1) a 5.67% weight loss upon drying 
at room temperature for 4 hours under vacuum, and (2) the composition 
47.68% carbon, 6.22% hydrogen, 11.48% nitrogen. These results are 
consistent with the empirical formula C.sub.11 H.sub.16 N.sub.2 O.sub.4 S. 
(NH.sub.3).sub.0.28, the calculated elemental composition corresponding to 
this empirical formula being C=47.68%; H=6.13%, N=11.52%, S=11.57% and 
O=23.1%. Polarimetric analysis of a 1 mg./ml. solution of this sample in 
10 mM potassium phosphate buffer showed a specific optical rotation 
[.alpha.].sub.D.sup.27.degree. C.+80. The infrared spectrum of a nujol 
mull of this sample revealed characteristic absorption peaks at 1765 
cm..sup.-1, 1650-1550 cm..sup.-1, 2800-2500 cm..sup.-1, and 3500-3100 
cm..sup.-1. An NMR spectrum at 100 MHz of a sample of this product 
dissolved in O.sub.2 O reveals a doublet at .delta.1.275, a pair of 
doublets at .delta.3.39 and multiplets at .delta.3.15 and .delta.4.20, 
these peaks being characteristic of thienamycin. 
The thienamycin obtained herewith may be acetylated according to the 
process of Example 8 to afford N-acetylthienamycin. 
EXAMPLE 9 
Acetylation of Thienamycin 
Thienamycin, 10.9 mg. is stirred for 10 minutes at 0.degree. C. in 1 ml. 
dry DMF plus 2 ml. freshly prepared acetic anhydride. The DMF and acetic 
anhydride are removed by washing repeatedly (5 to 6 times) with 25-40-ml. 
portion of hexane and one last portion of hexane after the addition of 1 
ml. dry ethyl ether. The crude sample of N-acetylthienamycin is dissolved 
in 20 ml. of deionized water which contains 100 .mu.moles of Tris base 
[tris(hydroxymethyl)aminomethane] and 35 .mu.moles of HCl. The pH, after 
dissolution of the sample, is 7.9. The solution contains 244 absorbance 
units at 298 nm and a 1/2 inch assay disc containing 0.1 ml. of a 1000 
fold dilution, produces a 23 mm. zone of inhibition when incubated on ATCC 
8461 plates at 25.degree. C. 
This sample is applied on a column (1.3 cm..times.14 cm. bed dimensions) of 
Dowex-1.times.4 (Cl.sup.-) minus 400 mesh. The column is washed with 10 
ml. of deionized water and the antibiotic, N-acetylthienamycin, is eluted 
with 0.07 M NaCl+0.005 M NH.sub.4 Cl+0.0001 M NH.sub.3 is deionized water. 
Fractions of 6.1 ml. are collected at a flow rate of 0.7 ml. per minute. 
The main peak of UV absorbance at 298 nm appears in fractions 36 through 
50, with a maximum at fraction 40. Fractions 38 through 46 are combined, 
containing a total of 107 absorbance units at 298 nm. The combined 
fractions are rotary evaporated under reduced pressure to 2 ml. and 5 
.mu.l of 1 M NaOH is added. 
This concentrate is applied on a column (2.2.times.80 cm. bed dimensions) 
of Bio-Gel P-2, 200-400 mesh. The sample is washed in with two, 1-ml. 
portions of deionized water and eluted with deionized water at a flow rate 
of 0.6 ml. per minute. Fractions of 3.04 ml. are collected. 
The main peak of UV absorbance at 300 nm appears in fractions 58 through 
64, with a maximum at fraction 60. Fractions 59 through 62 containing 83.7 
A.sub.300 units are pooled. A portion equivalent to 2.2 A.sub.300 units is 
removed for reference, and the remainder is concentrated to 1.5 ml. and 
lyophilized in a 14 ml. glass vial to give 3.9 mg. of N-acetylthienamycin. 
.lambda..sub.max 301 nm, E.sub.max /E.sub.min =4.45, E%.sub.301 in 
deionized water=208. 
Deacetylation of this starting material in accordance with the process of 
Example 2 affords the antibiotic thienamycin. 
EXAMPLE 10 
Preparation of Antibiotic 890A.sub.3 
A tube of lyophilized culture of Streptomyces flavogriseus MA-4434a is 
opened aseptically and the contents suspended in a tube containing 0.8 ml. 
of sterile Davis salts having the following composition: 
______________________________________ 
Davis Salts 
______________________________________ 
Sodium citrate 0.5 g. 
K.sub.2 HPO.sub.4 7.0 g. 
KH.sub.2 PO.sub.4 3.0 g. 
(NH.sub.4).sub.2 SO.sub.4 
1.0 g. 
MgSO.sub.4 . 7H.sub.2 O 
0.1 g. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
This suspension is used to inoculate four slants of Medium A having the 
following composition: 
______________________________________ 
Medium A 
______________________________________ 
Glycerol 20.0 g. 
Primary Yeast 5.0 g. 
Fish Meal 15.0 g. 
Distilled H.sub.2 O 1000 ml. 
Agar 20.0 g. 
pH adjusted to 7.2 using NaOH 
______________________________________ 
The inoculated slants are incubated for one week at 27.degree.-28.degree. 
C. and then stored at 4.degree.-6.degree. C. until used (not longer than 
21 days). 
Ten ml. of Medium B having the composition: 
______________________________________ 
Medium B 
______________________________________ 
Yeast Autolysate (Ardamine *) 
10.0 g. 
Glucose 10.0 g. 
Phosphate Buffer.sup. + 
2.0 ml. 
MgSO . H.sub.2 O 50 mg. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6.5 using HCl or NaOH 
______________________________________ 
*Ardamine: Yeast Products Corporation 
.sup.+ Phosphate Buffer solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 95.0 g. 
Distilled H.sub.2 O 1000 ml. 
is transferred aseptically to one of these slants, the spores and aerial 
mycelia scraped into suspension, and 3.3 ml. of this suspension used to 
inoculate a 2-liter baffled Erlenmeyer flask containing 500 ml. of Medium 
B. This seed flask is shaken at 28.degree. C. on a 160 rpm shaker (2" 
throw) for 36 hours at which time the growth is satisfactory. 
The growth from this seed flask is used to inoculate a 189-liter stainless 
steel seed tank containing 160 liters of Medium B. This tank is operated 
at 28.degree. C. using an agitation rate of 150 rpm and an airflow of 3 
cu. ft. per minute for 24 hours. Defoamer, Polyglycol 2000 (Dow Chemical 
Corp.), is used as required but not to exceed 0.1%. pH determinations are 
made as follows: 
______________________________________ 
Age, Hours 0 12 
______________________________________ 
pH 6.3 6.35 
______________________________________ 
Forty-three liters of the growth in this seed tank is used to inoculate a 
756-liter stainless steel fermentor containing 467 liters of Medium C, 
wherein Medium C has the composition: 
______________________________________ 
Medium C 
______________________________________ 
Tomato Paste 20.0 g. 
Primary Yeast 10.0 g. 
Dextrin (Amidex) 20.0 g. 
CoCl.sub.2 . 6H.sub.2 O 
5.0 mg. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7.2-7.4 using NaOH 
______________________________________ 
This tank is run at 25.degree. C. using an agitation rate of 146 rpm and an 
airflow of 9 cu. ft. per minute for 92 hours. Additional defoamer, 
Polyglycol 2000, is added as required, not to exceed a 0.1%. Antibacterial 
assays are run on Salmonella gallinarum MB-1287, Vibrio percolans ATCC 
8461 and the data is as follows: 
______________________________________ 
Age, Hours 
0 12 24 36 48 60 72 84 92 
______________________________________ 
pH 6.6 6.7 6.65 6.3 6.0 6.4 6.15 6.5 6.5 
MB-1287 mm. 
-- -- -- S -- 19 26 28 32 
ATCC 
3461 mm. 
(1-10) -- -- S -- 21 24 26 30 
890A 
units/ml. NA NA 6.8 13.5 24.9 24.3 
______________________________________ 
The 890A units/ml. are determined as set forth in Section II of Assay 
Procedures for Antibiotics 890A.sub.1 and 890A.sub.3. 
One hundred twenty-five gallons of broth is chilled to 5.degree. C. and 
centrifuged through a Titan P-9 centrifuge. Fifty pounds of Celite is 
added to the 100 gallons of supernatant, and the suspension filtered 
through a Shriver 18-inch filter press. The 91 gallons of filtrate is 
adsorbed on a column containing seven gallons of Dowex-1.times.2 
(Cl.sup.-), 50-100 mesh, and the column washed with 10 gallons of 
deionized water. The mixture of antibiotics 890A.sub.1 and 890A.sub.3 is 
eluted with thirty gallons of 5% NaCl+0.01 M Tris HCl buffer, pH 7.0+25 
.mu.M neutral EDTA in deionized water. Fractions of 5 gallons each are 
collected. The mixture of antibiotics 890A.sub.1 and 890A.sub.3 appears in 
fractions 3 through 6, with peak potency in fractions 4 and 5. Fractions 
4, 5, and 6, containing 8% of the activity of the filtered broth, are 
combined and concentrated under reduced pressure to 2 gallons. 
The two gallons of concentrate are applied to a column containing 10 
gallons of XAD-2 which had been previously washed with 50 gallons of 60% 
aqueous acetone followed by 50 gallons of deionized water and 50 gallons 
of 5% NaCl solution. The concentrate is eluted with 37.5 gallons of 
deionized water. Three fractions of 2.5 gallons followed by six fractions 
of five gallons are collected. The activity appears in fractions 1 through 
6, with a peak of potency in fraction 3. Fractions 4 and 5, containing 64% 
of the activity applied to the XAD column, or 370,000 units are pooled. 
Fractions 4 and 5 are concentrated to 120 ml. by evaporation under reduced 
pressure. The pH is adjusted to 6.5 and the concentrate is applied to a 
column (7.times.50 cm.) of XAD-2 which had been washed with 8 liters of 
60% aqueous acetone followed by 4 liters of deionized water and 8 liters 
of 5% NaCl in deionized water. The sample is drained to bed level and the 
column rinsed with three 20-ml. portions of deionized water, draining to 
bed level each time. The antibiotic is then eluted with seven liters of 
deionized water at a flow rate of 40 ml. per minute. Eight fractions of 
200 ml. followed by fourteen fractions of 400 ml. are collected. 
Antibiotic activity appears in fractions 4 through 19, containing 77% of 
the bioactivity applied on the column (as measured by the Salmonella 
gallinarum MB-1287 assay), with a peak of activity in fractions 6 through 
8. 
The ratio of bioactivity of the fractions on Vibrio percolans ATCC 8461 and 
the HAEA.sub.300 for the fractions is determined. Those fractions 
displaying the ratio value of about 250 indicate the presence of 
antibiotic 890A.sub.1 and those having a ratio of about 31 indicate the 
presence of antibiotic 890A.sub.3. Accordingly, fractions 10, 11, 12 and 
13, containing mostly antibiotic 890A.sub.3, are combined for further 
processing. 
Combined fractions 10 through 13 obtained from the second XAD-2 column are 
concentrated to 40 ml. and applied on a column (2.15 cm..times.40 cm. bed 
dimensions) of Dowex-1.times.4 (Cl.sup.-) minus 400 mesh. The antibiotic 
is eluted with three liters of 0.075 M NH.sub.4 Cl and 0.001 M NH.sub.3 in 
deionized water, at a flow rate of 3 ml. per minute. Fractions of 9 ml. 
are collected. Antibiotic activity, assayed by Vibrio percolans ATCC 8461, 
appears in fractions 165 through 234, with a peak of activity in fractions 
195 through 201. 
Fractions 186 through 213 are combined, containing a total of 472 A.sub.300 
units, of which 312 were extinguishable by reaction with hydroxylamine. 
These combined fractions are concentrated to 4.2 ml. by rotary evaporation 
under reduced pressure. The concentrate is applied to a column of Bio-Gel 
P-2 (200-400 mesh) with bed dimensions 2.15.times.60 cm. The sample is 
allowed to drain to bed level, and two rinses of 1 ml. each of deionized 
water are applied and drained to bed level. The column is eluted with 
deionized water at a flow rate of 1 ml. per minute, collecting fractions 
of 2.6 ml. each. 
The antibiotic 890A.sub.3 eluted in fractions 38 through 48, with a peak at 
fraction 41, determined by hydroxylamine-extinguishable absorbance at 300 
nm. Fractions 40 through 43 are pooled, containing 260 A.sub.300 units, of 
which 173 are hydroxylamine-extinguishable. 
To remove residual contamination of antibiotic 890A.sub.3 by antibiotic 
890A.sub.1, the combined desalted fractions 40 through 43 are treated 
wioth 50 .mu.l. of penicillinase [Difco "Bacto-Penase" containing 500,000 
units per ml. (1000 LU/ml. wherein the term LU refers to Levy units; 1,000 
LU will inactivate 500,000 units of penicillin G)] and 0.1 ml. of 1 M 
Tris-HCl buffer, pH 7.5. The reaction is allowed to stand at 23.degree. C. 
for 6 hours, and then 3 ml. of deionized water and 15 ml. of methanol is 
added. This mixture is applied on a column (2.15.times.44 cm. bed 
dimensions) of Dowex-1.times.2 (Cl.sup.-) minus 400 mesh, which had been 
packed in 50% methanol and washed with two liters of 50% (v/v) methanol. 
The antibiotic 890A.sub.3 is eluted with 2 liters of 0.05 M NaCl+0.005 M 
NH.sub.4 Cl+0.0001 M NH.sub.3 in 50% methanol. Fractions of 9.2 ml. are 
collected. The main peak of UV absorbance, measured at 300 nm, occurs in 
fractions 74 through 88. Fractions 78 through 85 are pooled. The total 
absorbance units at 300 nm in these fractions, after removal of the 
methanol by evaporation under reduced pressure, is 97.6, of which 87.5 are 
extinguishable by reaction with hydroxylamine. 
The pooled fractions 70 through 85 are concentrated to 3.92 ml. by rotary 
evaporation under reduced pressure, and the concentrate applied on a 
column of Sephadex G-10 (2.15.times.70 cm. bed dimensions) which had been 
washed with 4 ml. of 4 M NH.sub.3, followed by equilibration with 0.15 mM 
NH.sub.3 in deionized water. After two washes of 1 ml. each of 0.02 mM 
NH.sub.3, the antibiotic 890A.sub.3 is eluted with 0.02 mM NH.sub.3, at a 
flow rate of 0.8 ml. per minute. Forty-nine fractions of 2.45 ml., 
followed by twenty fractions of 3.33 ml. are collected. The main peak of 
absorbance at 300 nm appears in fractions 35 through 53. Fractions 38 
through 46, having the highest A.sub.300 /A.sub.245 values, are combined 
for further processing. The combined fraction have a total of 65 
absorbance units at 300 nm. The pooled fractions are rotary evaporated 
under reduced pressure to 2.84 ml., and divided into two equal portions 
which are quick-frozen and lyophilized separately in 14 ml. glass vials to 
give antibiotic 890A.sub.3. One sample contains 32.0 A.sub.300 units in 
0.88 mg., and the other contains 31.9 A.sub.300 units in 0.82 mg. The 
former sample containing 32.0 A.sub.300 units was subjected to NMR 
analysis and showed the following peaks: 
1.29 (d, J=6.5); 1.98 (s); 3.42 (d of d, J=5 and J=2.4); .about.4.01-4.28 
(m); 3.14 (d of d, J=5 and J=9); 3.39 (t, J=6.5); 2.92 (d of t, J=.about.4 
and J=6). 
The above table lists the 100 MHz-NMR signals for 890A.sub.3 sodium salt in 
D.sub.2 O relative to the internal standard, sodium 
2,2-dimethyl-2-silapentane-5-sulfonate; chemical shifts are given in ppm 
and coupling constants in Hz; apparent multiplications are indicated. 
Deacetylation of this material obtained herewith in accordance with the 
process of Example 4 affords the antibiotic desacetyl 890A.sub.3. 
EXAMPLE 11 
Preparation of Antibiotic 890A.sub.1 
A tube of lyophilized culture of Streptomyces flavogriseus MA-4600a is 
aseptically opened and the contents suspended in a tube containing 1.5 ml. 
of sterile Medium A having the following composition: 
______________________________________ 
Medium A 
______________________________________ 
Yeast Extract 10.0 g. 
Glucose 10.0 g. 
MgSO.sub.4 . 7H.sub.2 O 
0.05 g. 
Phosphate Buffer.sup.+ 2 ml. 
Distilled H.sub.2 O 1000 ml. 
______________________________________ 
.sup.+ Phosphate Buffer Solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 950 g. 
Distilled H.sub.2 O 1000 ml. 
This suspension is used to inoculate a 250-ml. triple-baffled Erlenmeyer 
seed flask containing 54 ml. of seed Medium B having the following 
composition: 
______________________________________ 
Medium B 
______________________________________ 
Autolyzed Yeast (Ardamine*) 
10.0 g. 
Glucose 10.0 g. 
MgSO.sub.4 . H.sub.2 O 0.05 g. 
Phosphate Buffer.sup. + 
2 ml. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 6.5 with NaOH 
______________________________________ 
*Ardamine: Yeast Products Corporation 
.sup.+ Phosphate Buffer Solution 
KH.sub.2 PO.sub.4 91.0 g. 
Na.sub.2 HPO.sub.4 950 g. 
Distilled H.sub.2 O 1000 ml. 
The seed flask is stoppered with cotton and shaken for 30 hours at 
28.degree. C..+-.1.degree. C. on a 220 rpm gyrotory shaker (2" throw). 
Fifty 250-ml. unbaffled Erlenmeyer production flasks, each containing 40 
ml. of production Medium C are inoculated with 1 ml. per flask of the 
broth from the seed flask. The production flasks are stoppered with 
cotton. 
______________________________________ 
Medium C 
______________________________________ 
Tomato Paste 20.0 g. 
Primary Yeast 10.0 g. 
Dextrin (Amidex) 20.0 g. 
CoCl.sub.2 . 6H.sub.2 O 
5.0 mg. 
Distilled H.sub.2 O 1000 ml. 
pH adjusted to 7.2-7.4 using NaOH 
______________________________________ 
After inoculation, the production flasks are incubated at 28.degree. 
C..+-.1.degree. C. with shaking on a 220 rpm gyrotory shaker (2" throw) 
for 3 days. The flasks are assayed for activity against standard Vibrio 
percolans ATCC 8461 assay plates using 1/2 inch assay discs dipped into 
centrifuged fermentation broth samples. Samples are diluted with 0.05 M 
phosphate buffer, pH 7.4. The results are tabulated below: 
______________________________________ 
Harvest Age Hours 72 
pH 6.4 
Vibrio percolans 
(1/100 Dilution) Assay 23 mm. 
890 Assay, units/ml. 103 
______________________________________ 
The 890A units/ml. are determined as set forth in Section II of Assay 
Procedures for Antibiotics 890A.sub.1 and 890A.sub.3. 
The whole broth is centrifuged in 200-ml. portions in polycarbonate bottles 
at 9000 rpm for 15 minutes to give 1600 ml. of combined supernatants with 
a potency of 104 units/ml. To this is added 0.5 ml. of neutral EDTA. 
The centrifuged broth is adsorbed on a Dowex-1.times.2 (Cl.sup.-), 50-100 
mesh column, bed dimensions 3.8.times.22 cm., at a flow rate of 6 to 20 
ml./min. The column is rinsed with 100 ml. of deionized water and eluted 
with 1 liter of deionized water containing 50 g. of sodium chloride, 0.02 
M Tris HCl buffer, pH 7.0, and 25 .mu.M neutral EDTA, at a flow rate of 6 
ml./min. Fractions of 10 ml. are collected. 
Antibiotic 890A.sub.1 appears in fractions 13 through 81, with a maximum at 
fractions 25 to 33, counting from the first application of salt eluate. 
Fractions 24 through 41, having the highest bipotency/A.sub.220 ratios, 
are combined for further processing. The combined fractions have a total 
of 29,000 units, or 17% of the applied bioactivity. 
The Dowex eluate is concentrated to 10 ml., the pH is adjusted to 6.5 with 
dilute hydrochloric acid, and the concentrate is applied on a column of 
XAD-2, bed dimensions 3.3.times.36 cm., which had been previously washed 
with 2 liters each of 60% aqueous acetone, deionized water, and 5% (w/v) 
sodium chloride in deionized water. The sample is eluted with deionized 
water at a flow rate of 6 ml./min. Fractions of 40 to 260 ml. are 
collected. 
Antibiotic activity appears in fractions 6 through 14, extending from 220 
to 2560 ml. of eluted volume. The peak is at fractions 9 and 10, extending 
from 370 to 550 ml. of eluted volume. Fractions 9 through 12, extending 
from 370 to 1060 ml. of eluted volume, have the highest ratios of 
HAEA.sub.300 /A.sub.220, and are combined for further processing. These 
fractions have 36,600 units, equal to 126% of the apparent applied 
activity. 
The combined fractions 9 through 12 are concentrated to 100 ml. and the 
concentrate applied on a column of Dowex-1.times.4 (Cl.sup.-), minus 400 
mesh, bed dimensions 2.2.times.41 cm., at a flow rate of 2 ml./min. The 
column is rinsed with 50 ml. of deionized water, and eluted with 3 liters 
of 0.07 M NaCl+0.005 M NH.sub.4 Cl+0.0001 M NH.sub.3 in deionized water, 
at a rate of 2 ml./min. Fractions of 10.8 ml. are collected starting from 
the first application of eluent. 
The main peak of antibiotic 890A.sub.1 appears in fractions 181 through 
217, with a maximum at fraction 198. Fractions 186 through 210, containing 
a total of 114 absorption units at 300 nm., are pooled. 
The pooled fractions are concentrated to 4.0 ml., and the pH is adjusted to 
7.3 by addition of 16.mu. liter of 1 M NaOH. The concentrate is applied on 
a column of Bio-Gel P-2, 200-400 mesh, bed dimension 2.15.times.70 cm., 
and is washed in with 3.times.1 ml. washes of deionized water and eluted 
with deionized water at 0.96 ml./min. Fractions of 3.85 ml. are collected. 
The main peak of antibiotic 890A.sub.1 appears in fractions 24 through 44, 
with a maximum at fractions 33 and 34. Fractions 27 through 38, having the 
highest A.sub.300 /A.sub.245 ratios, are combined for lyophilization. 
These combined fractions have a total of 72 A.sub.300 units. 
To carry out the lyophilization, the combined fractions are concentrated to 
3.0 ml. and the pH of the concentrate is adjusted to 7.5 by addition of 
10.mu. liters of 0.1 M NaOH. The sample is divided into two portions of 
1.50 ml. each, and the portions are separately quick-frozen and 
lyophilized from 14 ml. glass screw-cap vials. Each sample contains 1.73 
mg. of 890A.sub.1, corresponding to 35.8 A.sub.300 units. 
Deacetylation of this material obtained herewith according to the process 
of Example 3 affords the antibiotic desacetyl 890A.sub.1. 
Compositions containing desacetyl 890A.sub.1 and desacetyl 890A.sub.3, the 
antibiotics of this invention, and compositions containing thienamycin may 
be administered in several unit dosage forms as, for example, in solid or 
liquid orally ingestible dosage form. The composition per unit dosage, 
whether liquid or solid may contain from 0.1% to 99% of active material, 
the preferred range being from about 10 to 60%. The compositions will 
generally contain from about 25 mg. to about 1000 mg. by weight of the 
active ingredient based upon the total weight of the composition; however, 
in general, it is preferable to employ a dosage amount in the range of 
from about 250 mg. to 1000 mg. In parenteral administration the unit 
dosage is usually the pure compound in a slightly acidified sterile water 
solution or in the form of a soluble powder intended for solution. 
Representative formulations can be prepared by the following procedures: 
______________________________________ 
Capsules Per Capsule 
______________________________________ 
Desacetyl 890A.sub.1 400 mg. 
Lactose, U.S.P., a sufficient quantity to 
fill No. 0 Capsules, approx. 475 mg. each. 
______________________________________ 
In the above example the active compound and the diluent are mixed to 
produce a uniform blend, which is then filled into No. 0 hard gelatin 
capsules, by hand or on a suitable machine, as required. The mixing and 
filling is preferably done in an area having a relative humidity less than 
40%. 
______________________________________ 
Tablets Per Tablet 
______________________________________ 
Desacetyl 890A.sub.1 330. mg. 
Calcium phosphate 192. mg. 
Lactose, U.S.P. 190. mg. 
Cornstarch 80. mg. 
Magnesium stearate 8. mg. 
800. mg. 
______________________________________ 
In the above example, the active component is blended with the calcium 
phosphate, lactose and about half of the cornstarch. The mixture is 
granulated with a 15% cornstarch paste and rough-screened and screened 
again through No. 16 screens. The balance of the cornstarch and the 
magnesium stearate is added and the mixture is compressed into tablets, 
approximately 1/2" in diameter, each weighing 800 mg. 
Alternatively, the active component is blended with the calcium phosphate, 
lactose and one-half the cornstarch. The mixture is "slugged" on a heavy 
duty press to produce compacted tablet-like masses. These are broken down 
to a No. 16 mesh granule. The balance of the cornstarch and the magnesium 
stearate are added and the mixture is compressed into tablets 
approximately 1/2" in diameter, each weighing 800 mg. 
______________________________________ 
Lyo Form (For Injection) 
Per Vial 
______________________________________ 
Desacetyl 890A.sub.1 25 mg. 
Water-for-Injection, U.S.P. to make 
5 ml. 
______________________________________ 
In the above example the active component is dissolved in sufficient 
water-for-injection in the ratio shown. The solution is filtered through 
Selas candles or Millipore membrane filters to sterilize. The solution is 
subdivided into sterile vials. The vials and contents are frozen, and the 
water is aseptically removed by lyophilization. The vials containing the 
sterile dry solid are aseptically sealed. 
To restore for parenteral administration, 5 ml. of sterile 
water-for-injection is added to the contents of a vial. 
______________________________________ 
Oral Liquid Forms Per 1000 ml. 
______________________________________ 
Desacetyl 890A.sub.1 1.0 g. 
Sucrose 600.0 g. 
Glucose 250.0 g. 
Sodium Benzoate 1.0 g. 
Concentrated Orange Oil 
0.2 ml. 
Purified water U.S.P. to make 
1000.0 ml. 
______________________________________ 
The sucrose and glucose are dissolved in about 400 ml. of water using heat 
to aid solution. This solution is cooled and sodium benzoate, followed by 
the concentrated orange oil added. The solution is brought to about 900 
ml. volume with water and the antibiotic is added. The solution is 
clarified by filtration through a coarse filter.