JOURNMOFBIOSCIENCE ANDBIOENQINSBRINO

Vol. 91, No. 2, 123-128. 2001

Process Optimization for Enhanced Production of Diphtheria

Toxin by Submerged Cultivation
BHEEMAN SUNDARAN,‘* Y. UDAYA BHASKARA RAO,’ AND RATNAM BOOPATHY2
Pasteur Institute of India, Coonoor-643 IO3l and Department of Biotechnology & Enzyme Technology,
Bharathiar University, Coimbatore-641 046,2 Tamil Nadu, India

Received 28 July 2OOO/Accepted12 October 2QOO

When stationary culture was replaced by submerged cultivation ln a fermentor, a &n&ant increase ln the
yield of diphtheria toxin in a short cultlvatlon tlme (less than 48 h as against 7-g d) was noted. It was found that
under optlmal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin
release. Furthermore, to enhance the production volume, two-and three-step semlcontlnuous batch cultivations
were carried out. The toxin produced was of good titre wlth an adequate antigen& purity. Under optimal
conditions, the variation ln the Limes of flocculation (Lf tltre) was likely due to the quality of the produc-
tion medium, which in turn depended on the quaMy of the raw materials used. The process was also optimized
in a pilot-scale fennentor.

[gey words: Corynebacterium diphtheriae, submerged cultivation, fermentor, semicontinuous mode, anti-
genie purity, flocculation unit]

Diphtheria toxoid was one of the first immunization
agents introduced on a large scale in the form of the
triple vaccine comprising diphtheria, tetanus and pertussis
antigens. Mass immunization campaigns have made the
incidence of diphtheria extremely rare. However, the
true numbers of diphtheria cases and consequent deaths
are unknown because of incomplete reporting in most
countries where the disease is endemic. The World
Health Organization’s (WHO) recent estimate reveals
that there are about 100,000 cases worldwide and up to
8000 deaths per year (1). The same report also reveals
that the epidemics in different parts of the world are
largely due to decreased immunization of infants, waning
immunity to diphtheria in adults, movements of large
population groups and an irregular supply of vaccines.
In developing countries immunization was introduced in
late 1970s and the percentage of infants immunized with
three doses of diphtheria toxoid reached 46% in 1985
and 79% in 1992 (2). Thus, better control of the disease
depends on high immunization uptake.
Nearly all manufacturers employ a variant of the
Parke Williams 8 (PW8) strain of Cotynebucterium
diphtheriae to produce the exotoxin from which the tox-
oid is prepared by chemical modification (3). In general,
a medium formulation with amino acids, trace vitamins,
inorganic salts and a source of energy in the form of
carbohydrate promotes excellent growth characteristics
(4). Different media, such as gelatin-hydroiysate, the acid
digest of casein and the enzymatic digest (trypsin or
papain) of beef muscle, are found to be suitable for
toxin production. However, the most suitable medium is
the papain digest broth of beef muscle which consistently
gives a very high titre (5).
The simplest procedure for growth is the traditional
stationary culture method but this is labor intensive,
requires considerable space and is hazardous from the
viewpoint of sterility (6). Poor consistency in toxin yield
between consecutive production lots is generally ob-
* Corresponding author. e-mail: piicnr@md4.vsnl.net.in
phone: 0423-31250 fax: 0423-31655
123
served. Moreover, this type of culture is difficult to
scale-up and hence not economical. These inherent
problems can be overcome by adopting a submerged cui-
ture method in fermenters. In the present study, we have
used the papain digest broth of beef muscle along with
maltose as the sole carbon source for maximum toxin
production in a fermentor with a simple design and have
scaled up the culture to pilot level.
MATERIALS AND METHODS
Preparation of papain digest broth The required
quantity of minced beef muscle was suspended in sterile
water (1: 7 w/v) and 8 g of papain (Kumaon Nursery,
Nainital, U.P., India) as generally used to digest each kg
of beef. The digestion process was carried out as fol-
lows. The total quantity of the papain enzyme required
was weighed, powdered and suspended in 700ml of dis-
tilled water. Then lOOmi of this mixture was added to
the beef at 30 min intervals and the digestion was carried
out under controlled conditions of temperature (SO’C&
1’C) and pH (7.lfO.i). The duration of the digestion
was 3.5 h with adequate stirring throughout. Sodium
hydroxide solution (12.5 IV) was used for pH main-
tenance at 7.0-7.2. When digestion was complete, con-
centrated hydrochloric acid was added to reduce the pH
to 5.1 and the mixture was boiled for 10min. Glacial
acetic acid was added to reduce the pH to 4.1 and the
mixture was filtered using a cloth filter (7, 8). This broth
can be stored at 2-8°C for about 4 weeks without
significant loss in quality.
Iron concentration control and 6nal composition of
the basal medium Excess iron, which is detrimental
to release of the toxin, in the broth was removed by
treating it with baker’s yeast. In brief, the medium pH
was raised to 8.0 with 12.5 N sodium hydroxide solution
and yeast was added (4g/l). The temperature was main-
tained at 30-35X! for 60min while stirring. After 60min
the temperature was raised to 80°C and maintained for
1Omin to kill the yeast and the broth was clarified using
a cloth filter. This medium in which the iron concentra-
124 SUNDARAN ET AL.
tion was controlled was first clarified using a frame and
press-type filter with clarifying pads of the nonasbestos
type. One liter of the final medium contained 150 mg of
yeast extract, 1 ml of 60% sodium lactate solution, 1.5
ml of Mueller’s growth factor solution (4) and 300mg
of magnesium sulphate. Maltose at an initial concentration
of 2&0.5X was used as the energy source. The pH of
the medium was adjusted to 8.0 and the medium sterilized
by filtration (7, 8).
Starter preparation Throughout the study a sub-
strain of the classical PW 8 strain of Coryne&cterium
diphtheriae was used. The working seed lot was lyophi-
lized and stored at 2-8°C and revival of the seed was
performed on a Loeffler slope (7, 8): the culture for seed-
ing the laboratory-scale fermentor was obtained by in-
oculating the bacterium growing on a LoefRer slope into
250ml of production medium in a 1 I Erlenmeyer flask.
The flask was shaken at 14Orpm on a rotary shaker. To
seed the pilot-scale fermentor, 3 I of culture in a 10-I
flask were prepared. The duration of shaking was 20 h at
35°C. An inoculum of 1% was used to start the bioproc-
ess of toxin production.
Fermentor equipment The laboratory-scale (work-
ing volume of 25 r) and pilot scale (working volume of
35OI) vessels were composed of stainless steel. The de-
sign of the vessel is shown in Fig. 1 and the vessel is a
typical stirred tank type with six-blade turbine impellers.
The jars containing the sterile production medium were
placed in a water bath the temperature of which was con-
trolled at 35°C using a controller system. Conditioned
water flowed through the bath from a built-in recirculat-
ing water system equipped with a pump and an immer-
sion heater. The top plate harboured different ports as
well as the precision shafting box. Sterile air was passed
through a pressure regulator, flow meter and a re-
movable filter. The basic assembly of the latter unit in-
volves a moveable support with a variable drive and a
head plate with a stirrer shaft, bearings and seals. The
head plate also harboured industrial-type probes for
pH, dissolved oxygen and temperature measurement and
control. Compressed air of the required flow rate was
introduced through a steam sterilisable Domnic-Hunter
filter cartridge which was screwed into a special housing.
The control panel contained all valves for water, steam,
and gases, and the recorders, controllers and indicators
for temperature, pH and dissolved oxygen measurement
<TOP LID
GASKET
-SHAFT
/IMPELLERS
GASKET
d BOTTOM LID
FIG. 1. Schematic diagram of the laboratory-scale fermentor
which is a typical stirred tank type with six-blade turbine impellers.
The vessel is made up of stainless steel and the dimensions are: vessel
height, 45 cm; vessel diameter, 31 cm; shaft length, 37.5 cm; impeller
diameter, 11 cm.
J. BIOSCI.BIOFXNO.,
and control.
Culture conditions (a) Shaking cultivation was car-
ried out on a rotary shaker at 140rpm in 1 I Erlemneyer
flasks filled with 200ml of production medium. The
different carbon sources used were glucose, sucrose, dex-
trin and maltose. The flasks were seeded with a 1% in-
oculum and shaken for 48 h at 35°C. (b) The fermentors
containing sterile production medium were seeded with a
1% inoculum and cultivation was carried out at 35°C
for 40-48 h. The culture in the laboratory-scale fermen-
tor was agitated at 800 rpm and aerated at 3-4 1.min-l.
The agitation and aeration rates in the pilot-scale fermen-
tor were 6OOrpm and 14-16 I.min-I, respectively. Add-
ing 1Oml of silicone antifoaming agent to each jar effect-
ed foam control. In the pilot-scale fermentor 200ml of
the antifoam was used. Semicontinuous batch cultiva-
tions in the laboratory-scale fermentor were carried out
as outlined below. At the end of the first batch, the
biomass produced was removed and fresh medium was
added aseptically. The remaining culture (500 ml) was the
starter culture for the subsequent batch. In the same
way, step III cultivations were also carried out.
Medium analysis The cu-amino nitrogen content
was determined by the Sorensen titration method (9) and
the total nitrogen content was determined by the method
of Kjeldahl (10). Iron estimation was based on the mod-
ified Seamer method using bathophenanthroline (11).
Sodium dodecyl sulfate (SDS)-polyacrylamide gel (10%)
electrohoresis (PAGE) was carried out by the method of
Laemmli (12). High performance liquid chromatography
(HPLC) analysis was carried out using an analytical col-
umn (Protein-Pak 300 SW-Waters column with dimen-
sions of 30cmx7.5 mm id). The flow rate was kept at
1 ml/min using phosphate buffer pH at 7.2. The samples
were monitored at 210 nm.
Culture analysis Samples drawn at different time
intervals were subjected to the following tests: growth of
the organism was assessed based on visual comparison
of opacity unit between appropriate diluted culture sam-
ples and standard opacity tubes. From the observed
opacity unit the dry weight was calculated as described
by H. C. M. Brown (13). The other parameters analysed
were pH and flocculation units (14, 15).
Toxin analysis Protein nitrogen estimation for the
sterile toxin was carried out using the method of Kjel-
dahl (10). The antigenic purity was defined as the ratio
of the Lf unit to the protein nitrogen value and expres-
sed as Lf/mg PN (7).
RESULTS AND DISCUSSION
A steady increase in a-amino nitrogen content of the
papain digest was observed and a marked increase re-
sulted after boiling with hydrochloric acid at pH 5.1
upon the completion of enzyme hydrolysis indicative of
further degradation of the peptides. Treatment with gla-
cial acetic acid allowed the broth to be stored for longer
periods without deterioration in quality. Chemical ana-
lysis for the determination of the a-amino nitrogen, total
nitrogen and iron contents of the production medium
were carried out. The total nitrogen content was in the
range of 35O-4OOmg/lOOml. An a-amino nitrogen con-
tent of approximately 6Omg/lOOml and an iron content
in the range of 15-20 ,ng/lOOml consistently yielded a
high Lf titer (150-200). A low level of a-amino nitrogen
significantly reduced the Lf titer to <100 even in the
VOL. 91, 2001 PROCESS OPTIMIZATION FOR ENHANCED PRODUCTION 125

iron-controlled broth. Variations in toxin yield could be
attributed to diff&ences in the quality of the papain
batches used.
Stainer showed that toxoids prepared from beef broth
frequently contin antigenic material6 of bovine origin
(18). To identify any high-molecular-weight proteins in
the broth due to incomplete digestion, SDS ,PAGE and
HPLC were carried out. The results indicated the pres-
ence of low-molecular-weight substances (< 6000 daltons)
and hence the presence of any antigenic material of bo-
vine origin could be comple.tely ruled out. In addition,
the methodology used is iti compliance with WHO guide-
lines which stipulate that the digestion process is carried
out to ensure that the digestion has proceeded su&iently
to free the medium of extraneous substances (19).
Strains of C. diphtheriue ferment glucose, maltose
and galactose readily, whereas sucrose is unaffected.
Glucose in the broth at concentrations of 0.2% or more
acidifies it rapidly. Marked variations in the amount of
toxin produced were found in the case of fermentation
with dextrin. The likely chemical contaminants of com-
mercially available maltose are glucose and dextrin. The
effect of different carbon sources in the basal medium on
growth, pH and toxin yield was first investigated. The
different carbon sources used were maltose (2.5%), dex-
trin (2.5%), sucrose (2.5%) and a mixture of maltose
(1.5%) and glucose (1.0%). Shaking culture was em-
ployed and typical results are shown in Table 1. Growth
was very poor in the sucrose medium and no toxin was
detected at the end of the cultivation period.
The amount of release of dextrin glucose was low as
evidenced by the alkaline nature of the broth at the end
of cultivation. Growth was moderate (8,3 mg/ml dry
weight) and toxin secretion began at approximately 40 h
after the start of cultivation. The toxin quality was low
due to an unusually long flocculation time (kf). In mal-
tose medium, the pH dropped to near neutral, stabilized
during the toxin release and increased at the end of culti-
vation. A rapid drop in pH was observed in the broth
containing a mixture of maltose and glucose. The change
TABLE 1. Effect of different carbon sources on growth and toxin
titer in shaking culture
Sampling time (h)
Carbon source Parameters
A 18 94
in pH was due to the production of organic acids from
readily available glucose in the broth. The extent of
growth obseived was equal to that in the broth contain-
ing maltose as the sole carbon source (19.2 mg/ml dry
weight). Toxin synthesis was affected by the low pH as
reported by Van Hemert (Thesis, Delft, 1971). He ob-
served that the course of pH in a culture exhibiting low
toxin production (toxin yield) differs distinctly from the
pH curve for a culture exhibiting normal toxin produc-
tion. Maltpse was found to be the most ideal carbon
source for commercial toxin production. Moreover, pH
maintenance in the broth containing glucose necessitated
the use of sophisticated pH control equipment, whereas
the use of maltose medium simplifies the fermentor
design which was the main aim of this study.
Having optimized the carbon source, growth and tox-
in production were compared between shaking culture
and fermentor culture (batch mode) utilizing maltose as
the sole carbon source. Growth and toxin production
were significantly higher in the fermentor culture than in
the shaking culture when the cultivation time was con-
stant (Fig. 2a, b). The composition of the basal medium
was the same for both cultivation methods. Shaking
culture was carried out on a rotary shaker at 140 rpm.
The fermentor culture was agitated at a shaft speed of
0 6 12 18 24 30 36 42 48
Sampling time (h)
Al7 AR

PH 1.8 1.45 1.55 1.9 8.0

Maltose 2.5% Growths 0.28 6.9 8.3 16.6 19.2
Lf titer ND Nil 20 50 60
Flocculationthue (kf)inmin - - 5 2 2
PH 7.6 8.0 8.1 8.9 9.1
Growth’ 0.14 5.5 6.9 8.3 8.3
Dextrin 2.5%
Lf titer ND Nil Nil 20 25
Flocculation time(kf)inmin - - - 15 30
PH 7.9 8.2 8.3 8.5 8.55 1 I I 1 . .
Sucrose 2.5% Growth” 0.14 2.8 4.1 5.5 5.5 0 6 12 16 24 3b 36 42
Lf titer ND Nil Nil Nil Nil
Sampling time (h)
Flocculationtime inmin - - - - -
6.9 5.3 5.35 5.4 5.42 FIG. 2. (a) Toxin production was expressed as flocculation units
PH
Maltose 1.5% & Growth” 0.28 6.9 8.3 16.6 19.2 (Lf/ml) Sampling was done at different hours and the Lf titer in
Glucose 1.O?$ Lf titer ND Nil Nil Nil Nil fermentor culture (+) and in shaking culture ( n ) was determined as
Flocculationtime(kf)inmin - - - - - described in Materials and Methods. (b) Growth prolile of C. dip-
theriue during cultivation in the shaking flasks ( l ) and in the fer-
B Dry weight calculated based on the opacity table and expressed in mentor (+). Sampling was done at different hours and the opacity of
mg/ml(13). the culture samples were visually compared after appropriate dilution
ND, Not done. with physiological saline, with standard opacity tubes and the dry
Nil, No toxin. weight was calculated.
126 SUNDARAN ET AL. J. BIOSCI.BIOENG.,

800rpm and well aerated at 34 Z.min-i. The growth als and Methods) was tried. The operational conditions
rate was extremely high in the fermentor culture and a were the same as those for batch mode cultivation. A
biomass of 33.2 mg/ml dry weight was obtained which consistently high Lf titer was obtained in two-step as
was almost twice that of the dry weight obtained per ml well as three-step cultivations. The antigenic purity of
in the shaking culture. In addition, the toxin yield was 3 the sterile toxin was >lOOOLf/mg PN. Table 2 gives
times higher in the fermentor than in the shaking culture typical results obtained in five trials in which the titer
per unit volume. In the laboratory-scale fermentor, the was high and adequate purity signified that the cells did
pH profile was characterized by an initial drop from 7.8 not undergo any degenerative changes but preserved their
to approximately 7.0 and then plateaued between 7.2-7.4 morphology and doubling capacity.
during which time active synthesis and release of the Semicontinaous three-step cultivation technique
exotoxin to the medium were observed. According to Batch mode cultivation was routinely used to produce a
Tasman and Brandwijk, acid formation in a maltose biomass of 75 I. Using the semicontinuous process, 150 1
medium is slow since the splitting of this disaccharide is or 225 1 of biomass were produced in two-step and three-
the limiting step (21). The agitation and aeration rates step cultivations, respectively, over a period of one
are the main parameters affecting the oxidation of carbox- week. The semicontinuous mode was more economical
ylic acids produced during fermentation. than the single batch mode since the three-step cultiva-
Subsequent to the standardization of operational con- tion could be completed in 6 working days with only one
ditions in the batch mode cultivation in the laboratory- set of preparations and one sterilization and washing of
scale fermentor (using the pH protile as an index of max- the cultivation jars.
imal yield), maltose from four different sources of sup- The next experiment was to scale up the production
ply were tested. Maltose batch I, which was procured from using a pilot-scale fermentor. The medium composition
an indigenous source, did not induce the production of used was similar to the one used for the laboratory-scale
toxin eventhough the growth of the bacterium was ade- fermentor. After trial and error, operational parameters
quate. Maltose batches II, III and IV, which were im- such as the aeration and agitation rates were stan-
ported with the assistance from UNICEF, resulted in dardized for 300 I of biomass production so as to main-
high titers (180-200). A rapid drop in pH was noticed tain the typical pH profile. As can be seen from Fig. 3a,
in the culture containing maltose batch I. Impurities in the on-line data of pH coincided quite well with the
the maltose preparation might be the reason for this off-line data. The pH value leveled off between 7.2-7.4
problem. On chemical analysis, the maltose purity of
batch I was found to be 80%, whereas that of the other
batches were >98% pure (unpublished data).
A set of experiments using different initial concentra-
tions of maltose, viz 2.5%, 2.0% and l.S%, was carried
out to evaluate the effect of the initial concentration of
carbon source on toxin yield. The biomass production
was significantly high for the highest concentration of
maltose, but the toxin yield was practically the same as
was the pH at the end of the fermentation period. It is
evident that growth rate and toxin yield are not related.
Economic utilization of expensive maltose can be exer- 6 7-
cised when large-scale cultivation is carried out.
To augment the biomass production, a semicontinu- 6.8, I I I I I I I I I t I
ous mode of batch cultivation (as described in the Materi- 0 4 8 12 16 20 24 28 32 36 40’ 4
Sampling time (h)
TABLE 2. Growth rate, pH, toxin yield and antigenic purity in
semicontinuous mode cultivation (three steps) in the
laboratory-scale fermentor
Value at 42-46 h
Mode Antigenic purity
(Lf/mg PN)
1 7.5 4 190 1248
2 7.6 4 150 1275
STEP I 3 7.4 4 z 1470
4 8.1 5 1275
5 7.6 5 190 1380

: 76
8’1 5 200 1291
1275
V. I I , I I I I 9 I . 9

STEP II 3 7:6 7 1123

0 4 8 12 16 20 24 28 32 3840 44
4 8.0 5 250 1291
5 8.0 5 260 1428 Sampling time (h)

1 7.9 5 210 1008 FIG. 3. (a) On-line measurement of pH during cultivation in the
2 8.0 6 220 1165 pilot-scale fermentor. The pH was measured with the help of an
STEP III 3 7.5 6 200 1165 industrial-type sterilizable electrode (Ingold). (b) On-line measure-
4 7.9 5 300 1275 ment of dissolved oxygen @Oa) during cultivation in the pilot-scale
5 7.9 6 300 1284 fermentor. The pOz measurement was possible with the help of an
industrial-type sterilizable electrode (Ingold).
 PROCESS OPTIMIZATION FOR ENHANCED PRODUCTION 127

TABLE 3. Comparison of antigenic purity of crude toxin

produced by static and fermentor cultivation techniques

Culture Duration of Lf titer at

Method of Antigenic
volume cultivation the end of
cultivation puritya
ln (h) growth cvcle”
Static 8 168 80 700
Fermentor 15 42-46 180 1200
a Average value of five independent trials.

In Conclusion to keep diphtheria under control, a

sufficiently high percentage of the susceptible population
must be immunized and this immunity must be rein-
1 2 3 4 5 6 8 9 10 forced by repeat doses. To prepare toxin to meet the
Trials demand, the toxin produced per unit volume of the me-
FIG. 4. Toxin yield in the pilot-scale fermentor. Each batch was dium must be increased. By adopting fermentor technol-
used for 10 trials and the Lf titer was performed on the samples drawn ogy, as described in this study, larger production batches
at the end of cultivation as described in the Materials and Methods. are possible. The amount of toxin required will deter-
Symbols: 0, maltose batch I; w, maltose batch II. mine the vessel capacity. The use of a fermentor with a
simple in design proved to be an economical means of
during active synthesis and release of toxin to the broth. producing unlimited quantities of toxin. A measurable
In addition the on-line data showed that the pOZ value reduction in the production cost will lead to better use
dropped to 10% and was maintained at this level during of resources for health particularly in developing coun-
the toxin production stage (Fig. 3b). According to Van tries. We consider this type of versatile laboratory-scale
Hemert, the oxygen supply could be considered adequate fermentor to be ideally suited to the needs of developing
when the pH is between 7 and 8. countries where the National Immunization Programs
Two maltose batches obtained from two sources were often face exceptional challenges in the form of short-
each used for 10 trials. The initial pH was kept at ages of vaccines. To set up a production facility, the
7.9kO.l and a consistently high Lf titer of >200 was assistance of UNICEF can be sought since it plays a cri-
obtained (Fig. 4). The antigenic purity of the sterile toxin tical role in the acceleration of immunization activities.
was in the range of lOOO-1200Lf/mg PN (unpublished Furthermore, the fermentor design can be adopted for
data). Toxoids synthesized from these batches, after the production of other components such as pertussis
purification, had titers higher than 1500Lf/mg PN and tetanus toxins (aeration is not required). Such inter-
which is the minimal requirement according to WHO changeability of function is the “major advantage” of
guidelines (7, 8). benefit from this cultivation system.
Successful toxin production depended on various fac-
tors such as the design of the fermentor, proper agita- ACKNOWLEDGMENTS
tion, aeration and the quality of the raw ingredients in
the medium. Based on our results, it was evident that The authors would like to thank Mrs. Rohini Gideon (Research
Officer-Retd), Dr. A. K. Thomas (Technical advisor and former
the level of a-amino nitrogen, the iron content and the
Director of Central Research Institute, Kasauli) and Dr. V. R.
purity of the carbon source could indicate the quality of Kalyanaraman (Former Director of the Pasteur Institute of India,
the production medium. Iron control in the production Coonoor) for their encouraging support and valuable technical
medium is crucial as reported in previous studies (16, discussion. The technical assistance of the staff of the Diphtheria
17). The medium is heat labile and sterilization by filtra- Production Lab is greatly acknowledged. The authors acknowledge
tion must be practiced. Furthermore, to ensure a high the technical assistance of Mr. Vasudevan (J.S.S. College of Phar-
degree of quality, the main chemicals, such as maltose, macy, Udhagamandalam) in carrying out the HPLC analysis of the
papain, and baker’s yeast must be procured in large production medium. They also acknowledge the assistance of Mrs.
quantities from tested batches. Optimising the culture Rajam Muralidharan (Librarian, P.I.I.C.), Mr. P. P. Padmana-
bhan (Bharathiar University, Coimbatore) and Mrs. J. Sunitha
parameters compensated for of variations resulting from
Raju (Enterovirus Lab, P.I.I.C.) in preparing the manuscript.
differences in the quality of the raw materials to a great
extent. For diphtheria toxin production, the basic re-
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