Manufacturing Process
A culture of NRRL 2773 is produced by growing the organism on a nutrient
agar slant having the following composition:
Oatmeal-Tomato Paste Agar
Tomato paste 20Grams
Precooked oatmeal 20Grams
Agar 15Grams
Tap water, added to make a final volume of 1 liter.
The slant is inoculated with spores of NRRL 2773 and is incubated for 10 days
at about 30°C. The culture growth on the slant is covered with 6 ml of
nutrient broth, and the slant is scraped gently to remove the organisms to
provide an aqueous suspension. Employing aseptic techniques, the inoculum
obtained from one 1-inch agar slant is used to inoculate a 2-liter Erlenmeyer
flask containing a 500-ml portion of a sterilized vegetative culture medium
having the following composition: soluble starch, 10 g; peptones, 5 g; beef
extract, 5 g; sodium chloride, 5 g; yeast extract, 2.5 g; and tap water, 1,100
ml. The incubation is carried on at 28°C for 48 hours with shaking at 250
cycles per minute on a rotary shaker having a 1-inch stroke.
To produce a larger quantity of vegetative inoculum, 500 ml of the vegetative
inoculum is added aseptically to a stainless steel 350-gallon fermentation tank
containing 250 gallons of sterile medium having the following composition
(weight/volume): glucose, 1.5%; yeast, 1.5%; and antifoam (Polyglycol No.
2000, Dow Chemical Co.), 0.02%. The inoculum is allowed to grow for about
22 hours at a temperature of 30°C. Throughout the growth period, the
medium is aerated with sterile air at the rate of 17 cfm and is agitated with
two 16-inch impellers rotating at 160 revolutions per minute. To a 1,700-
gallon stainless steel fermentor are added 1,100 gallons of a medium having
the following composition (weight/volume):
Peptone No. 159 Medium
Glucose 2.5%
Molasses 1.0%
Peptones 4.0%
Calcium carbonate 0.2%
Hydrolyzed casein 0.6%
Antifoam (Polyglycol No. 2000, Dow Chemical Co.) 0.005
The medium after sterilization is inoculated with 100 gallons of the inoculum
grown in the fermentation tank. The fermentation is carried on at 30°C for
about five days. The foam is controlled by the addition, when needed, of
Larex No. 1 (an antifoam product, Swift and Co.). Throughout the
fermentation, the medium is aerated by the addition of sterile air at the rate
of 96 cfm and is agitated with two 22-inch impellers operated at 140
revolutions per minute. At the end of the fermentation, 240 lb of Dicalite 476
(a perlite filter product, Great Lakes Carbon Corporation) are added to 1,000
gallons of the antibiotic broth, and the mixture is stirred and filtered. The filter
cake is washed with tap water and the wash water and the filtrate are
combined to provide a total volume of 1,000 gallons.
To 500 gallons of the combined liquids are added 132 lb of Darco G-60. The
mixture is stirred thoroughly and filtered, and the filtrate is discarded, The
carbon filter cake is washed with 200 liters of tap water, the wash water being
discarded. The washed carbon cake on which the capreomycin is adsorbed is
further washed with 200 liters of 0.05 N aqueous hydrochloric acid. The acid
wash is discarded. The washed carbon cake is eluted during a one-hour period
with 400 liters of an aqueous acetone mixture containing 1.65 liters of 11.7 N
hydrochloric acid and 80 liters of acetone. The filter cake is further eluted by
washing the cake with 200 liters of an aqueous acetone mixture containing
825 ml of 11.7 N hydrochloric acid and 40 liters of acetone during a 15-
minute period. The combined eluates, having a total volume of 575 liters, are
concentrated in vacuo to 52.5 liters.
The concentrate is added with stirring to 525 liters of acetone and the acetone
mixture is permitted to stand overnight at room temperature, during which
time an oily precipitate of capreomycin separates. The supernatant is
decanted and discarded, and the oily precipitate which remains is dissolved in
20 liters of distilled water. The aqueous solution is concentrated in vacuo to 12
liters to remove any residual acetone. The aqueous concentrate containing
capreomycin is filtered to remove a small amount of a precipitate, which is
discarded.
The filtrate containing the capreomycin is added to 240 liters of methanol with
stirring. The methanolic solution of capreomycin is acidified by the addition of
one liter of 10 N sulfuric acid, whereupon the precipitation of the sulfuric acid
addition salt of capreomycin commences. The mixture is permitted to stand
overnight for more complete precipitation. The supernatant is removed by
decanting and filtering. The precipitate, consisting of the capreomycin
disulfate, is washed with 10 liters of methanol and is dried in vacuo. Yield:
2,510 grams.
References
[1] MARCOS ABDO ARBEX. Antituberculosis drugs: drug interactions, adverse effects, and use in special situations. Part 2: second line drugs.[J]. Jornal Brasileiro De Pneumologia, 2010, 36 5: 641-656. DOI:
10.1590/s1806-37132010000500017[2] VIANNA J F, BEZERRA K S, OLIVEIRA J I N, et al. Binding energies of the drugs capreomycin and streptomycin in complex with tuberculosis bacterial ribosome subunits[J]. Physical Chemistry Chemical Physics, 2019, 35: 19192-19200. DOI:
10.1039/c9cp03631h[3] W. B. SUTTON. IN VITRO AND IN VIVO LABORATORY STUDIES ON THE ANTITUBERCULOUS ACTIVITY OF CAPREOMYCIN[J]. Annals of the New York Academy of Sciences, 1966, 135 2: 947-959. DOI:
10.1111/j.1749-6632.1966.tb45536.x
