Journa l of Scientific & Industrial Research

Vol. 60, October 200 I, pp 773-778

Large Scale Processing of Tetanus Toxin from Fermentation Broth

S D Ra vetkar ' , S 8 Rah alkar and C G Kulkarni
Serum Institut e of I ndia Research Found:1tion, 2 12/2, Hadapsar, Punc 4 11 02~. India

Ph: lJ 1-20-6993900: fax: lJ 1-20-6993921

Received: I Jan uary 200 I : accepted: 2 1 June 200 I

Separation of bacterial ce ll s, conccntrat ion and stcri lc fi I ration arc some of the m:1jor unit operations in th e processing of large
volu mes o f ferment at ion brot h in the producti on of bact erin ! vaccines. Separation of cells is traditionall y :Jchicvcd by ccn tri fugation
or by dead-ended depth filtrat ion. These so lid-liquid scpMationmcthods arc time consuming, tox in recoveries ;lrc not sat i sfactory
and it is diffi cult to validate a depth filtration sys tem. Tctnnu s toxoid i s prepared by detoxifying th e cu lt ure filtrates of Clostridiu111
teta11i :1nd further purifyin g it by ultrafi ltrntion and s;1 lt fr;1ctinnntio n. This nrtic lc desc ribes compa ri son of performance of dead-
end filt rat ion and Tan gentia l Flow Filtration (TFf<) system for cln rifi c;l ti on of tct;mus fcrmcnt :Jt ion broth and furth er usc of ;1
simi l:~r system forconccntr:J tion ;111d purification oftctnnu s toxoid .

in th e processing of large volumes of fe rmentat ion media

Introdu ct ion
Immuni zat ion is a signifi ca nt compo nent o f Prim ary
Hea lth Care and late ly lot of emph as is has been placed
on immuni zat ion as a hea lth inte rve nti o n and has
ultimately tak en the shape of Uni ve rsal lmmuni za ti on
Prog ram. DPT (diphth e ria , pertu ss is and tetanu s) is
co mbinati on vacc in es, whi ch hold brig ht fu ture for
immuni zat ion prog ram . DPT group of vacc ines form a
major part or vacci nes again st th e "targe t" d iseases
identifi ed by th e immuni zati on program. Presentl y, th ere
are several manufacturers of OPT group or vacc ines. lt
sho uld be rea li zed tha t th e tec hn ology o r vaccine
prod uction is changing at a fa st pace and . th e refore, the
ex istin g units should ge nerat e enough surp lu s to in vest
in R&D- not onl y fo r products a lone , but fo r th e
manufacturin g meth ods also.
One area o f deve lopme nt s is to produce a purer
produc t. l t w ill red uce th e incid e nces or unt owa rd
reacti ons, after immuni zation , as th e react ions are oft e n
caused by th e presence of non-specifie mate rial s. i e.
impurities. Purer anti ge ns would also res ult in better
qua lity co mbin ati on vacc in es with less sup rressive
interacti on with oth er anti ge ns.
Separation of bacterial ce ll s, concentrat ion and :-; tcri le
filtrat ion of tox ins are so me of the major uni t ope rati ons
· CorrcsronJi ng author
in th e prod ucti on of bacte ri al vacc ines. The separati on
or cell s is th e most importan t step, whi c h is achi eved
traditi onall y by ccntri fu gati on or by the dead-ended dept h
filtra tion meth od. Such so li d- li quid sepa rati on methods
a re tim e con sumin g, and toxin recove ri es a re not
sati sfactory. Furt herm ore, it is diffi cult to va lidate a dept h
filtrati on system. whi ch is manda tory und er GMP (good
manufac turin g pract ices).
Me mbrane filters sepa rat e com po ne nts and
suspe ns ions on the bas is of s ize. In ton gcntiol.flmr
.filtmtion (TFF) mode, particles or so lutes retain ed by
me mbran e arc con tinu ously re moved in th e retcn tat e
fl ow ing tangentia ll y across the me mbran e surface. The
clarified so luti on flows through th e membrane int o the
pe rmeate, also ca ll ed th e cmssflowflltmtio n .
Tetanu s toxo id is prepared by detoxify in g the cultu re
filt rates of Clostridiu111 tetoni, and furth er purifyin g it
by ultra filtrat ion and sa lt fractionatio n. Sati sfactory
remova l of ce ll debris (i e, clar ifi ca ti on) is criti ca l to
obtain good yiclds.lnthe co nventi onal met hod . the dead-
end filtrati on, incorpo ratin g filter pads or sui table pore
sizes, th e sepa rati on must pro vi de co mpl ete rete ntion or
ce ll s, and maximal passage and recove ry of so lu ble end-
product . Whe n tetanu s ferme nt ati on broth is clarified.
th e large bac terial mass bloc ks mos t of the dead ended
filtration pads req uirin g still hi gher inl et pressures or
change of pads altogeth er. The cla ri fi cd tox in so lution is
774 J SCII ND RES VOL 60 OCTOBER 2001
then polished by pass in g it through pad filters to produce
sterile tetanus tox in . Also, the depth filtration is not a
closed process (due to the need to change pads during
the process) and the working perso nnel are exposed to
the toxin. Additionally, the pads also absorb the active
material, resultin g in reduced yields.
Another GMP aspect observed is the inab ility to tes t
integrity of pads and poss ible leachin g of ex tractabl e
matter. For th ese reasons, an alternative tec hn ology was
looked at seriou sly, th at could be validated, and the trial s
were conducted.
A mi croporou s TFF sys tem provides a practi ca l an d
economi ca l alternative to the dead ended filtrati on 1 • It is
a closed system which reduces the exposure of personnel
to the tetanus culture. The entire operation conform s to
th e GMP norm s and can be validated as and when
required 2 . Validation is a key step in th e ove rall
manufac turing process. It is defined as the procedure of
obtaining and documenting evidence of the performan ce
of a system, which adequately demonstrates compliance
with the design criteria.
The study compares the conventional depth filtrati on
method with that of mi croporous tan genti al fl ow filtration
(TFF), es tabli shes parameters th at opt imi ze overa ll
concentrat ion, di afiltration and result in impro vement
in the preparation of tetanus vaccine. It also desc ribes
aspects of validation of th e TFF system.
Materials and Methods
The Harvard strain of Clostridium tetani (from the
Central Research Institute, Kasauli , India, th e national
control authority) was used for th e producti on of tetanus
toxin in fermenter vessels ( 1000 L). Muell er and Miller
(M&M) medium' was used for growin g C/. tetani . The
fermentation vessel had a workin g capacity of I000 L.
Sterilization of the med ium was carried out by steam at
12 I oc for 30 min and after cooling it down to 35 oc, it
was inoculated with a I d old seed and incubated for 6 d
at 35 oc under co ntinu ous mild agitat ion.
At the end of the incubation period, samp les were
drawn for purity check s and estimati on of anti ge n
con ce ntration. Th e purit y was c hecked by : ( i)
microscopic observation, wherein on ly the Gram-posit ive
bacill i should be visible, (ii ) by inoculatin g nutri ent agar
broth/slants and incubatin g th ese fo r 2 d, wherein no
growth shou ld be visible.
The ant ige n concentrati on was determin ed by th e
cla ss ical floc culati on meth od (Ram on Fl occ uiJti on)
wherein the sa mpl e is fl occulated again st a standard
antitoxi n of known strength (Lf/mL).
If th e fermented broth is fou nd to be pure and of
sati sfactory concent rat ion of the tox in (Lf/mL ), it is
subj ec ted to further filtration and processin g as detail ed
below.
A: Dead-Ended Depth Filtration
Filter Mat erial
Filter pad s, 20 em x 20 em , 3-5 11 pore size (K- 200
pads, Seitz Filter Werke Bad Kreuznach , West Germany);
Filter Press Assemblies (S traussburger, West Germany)
The press assemb ly holds 24 pads.
Operating Conditions
The operati ons were carri ed out at 34-35 oc und er I 2
ps1 pressure.
Performance
The output was 80-100 L filtrate in 50-60 min and
required 300-400 filter pads to filter 1000 L broth. The
output thu s obtained was subjected to another depth
filtration using E KS pads, 20 x 20 em, and 24 pads were
required to obta in a steril e prod uct.
The procedure detai led required chan ge of pads during
the process. A sli ght variation in inlet pressure results in
a turbid output, needin g yet anoth er clari fication cyc le.
Thi s problem was encountered in 18 ou t of 52 batches
processed in a year.
B: Tangential Flow Filtration
It is essenti al to select a su itab le TFF dev ice taking
into account the criti ca l aspects o f ce ll re cove ry,
possibility of shearin g of cell s within th e dev ice, control
of temperature, repeated usage, possibility of steami ng,
etc. fn order to find a suitab le soluti on to above problems,
a set of trials was co nducted on a pil ot unit of TFF system
(Mi ll ipo re Ltd ) wi th hydrophi lic PVDF membran e of
0.22 ~~p o re si ze. whi ch has a very low prote in bi nding
characteristi cs. It co nstituted th e vali datab le devi ce for
biopha rm aceut ical ap plicati ons .
Fi ve batches we re co mpared with cla rification sluui es
on Pros tak Pilot unit. Subsequent ly, production scale
ba tch es we re tak e n up and th e sy st e m wa s we ll
standardi zed for I 000 L batches.
 RAVETKAR 1'1 a/.: PROCESS I G OF TETANUS TOX IN
System Description
'M illipore Pros tak Demo Unit ' was selec ted for the
cla rifi cati on of tetanus toxoid broth . The unit has a screw
pump con nected to a variab le drive. Flow rate of the
pump can be varied from 0 to 30 Llmin. A pressure gauge
was in stalled on the feed line and another one on th e
retentate line. A sanitary type diaphragm valve was also
installed on the retentate line, whi ch is used in controlling
the trans- membrane pressure, ac ross the membrane.
Prostak modules (Catalogue o PSDVAG021.
0.22f.lm, 2 ft 2 ) were used for the trials. Five modul es
were installed in a series. The feed ente red th e first
modul e through the feed manifo ld of th e Prostak holder
and retentate of the first modul e entered as a feed to th e
second module . The arrangement conti nued and th e final
retentate ca me out from the fifth modul e. The permeate
from first modul e entered into the permeate chann el of
the second modul e, and from second to third it flows up
to the fifth modul e. It is co ll ected after the fift h module .
The trials were conducted to remove cell debris from
fermenta tion broth and sim ultaneously clari fying th e
tetanus toxin using Durapore Prosta h: Modu le of O.n p
pore size. The modules we re se lected for the ce ll
clarification step due to their open channel design as there
would be hi gher chances of membrane fouling in case
of thin cha nn e l design. For the pi lot studi es, I 00 L
cultures, each from five successive batc hes , we re used
for filtration on pilot- scale for TFF system. The to xin
was processed throug h the Prostak mod ul es. Diafiltration
was clone with normal sa line. Five 20ft" mod ules were
used for trials and th e syste m was ope rated at a cross
flow of 2000 L/h at an operating temperature of n C.
The operating paramete rs were optimi zed based on rl ow
and pressure excursion experiments .
After the filtration of the toxin was comp lete, the
furt her processing involved detoxifi ca tion , and 0.45 per
cent (v/v) fo rm alin was added for the purpose, and after
adjusting pH to 7.6±0.5 , it was incubated at 35±1 oc for
28 d. After completion of detoxification , the toxoid was
checked for its spec ific toxicity. Once the toxoid passed
the specific toxicity test, it was concentrated usin g the
Pell icon system, described subsequently.
Valida tion of the TFF System
The selection of a membran e, as specified earlier. was
mainly guided by the possibility of validat ion of the
775
sys te m . Validation of th e system includ ed: ( i)
determination of th e pre- and post-operation integ rity
testing of the membrane dev ice, which ensured repeated
use, (ii ) removal of th e storage agent, i e, fo rm alin from
the modul es. Fo rm alin was removed by flushin g the
system with distilled water. Samples were co llec ted at
different intervals. To 3 mL sa mpl e, 0.5 mL chromotropic
acid was added and the mi xture was placed in boiling
water bath fo r 5 min. The prese nce of formalin was
co nfirm ed by th e deve lopme nt of pink co lour, (iii )
check ing absence of C/. tetoni- as th e membrane used
was of pore size 0.22 p , the absence of C/. tetoni from
the c larifi ed tox in needed to be va lidated. Th is was
carri ed out by passing a rep resentative samp le of 250
mL through the GVWP disc (Durapore PVDF 0.22~t
disc), whi c h was placed on th e surface of M&M agar.
On anoth er M & M aga r plate, overnig ht grown culture
of C/. tetani in fluid thioglycollate medi um was streaked
as a positive co ntroL (iv) sanitization of th e sys tem-
for sani ti zin g th e system, chemica ls such as acetic acid
a nd sod ium hypochlorite \vcre used . The cleaning
procedure ensured re moval of th ese c lean in g agents.
Removal of aceti c acid (below I0 ppm) was confirmed
by comparin g seriall y d iluted sample of acetic acid with
methyl reel indi cator. In case of sodium hypochlorite.
procedure similar to that ad opted in case of acetic ac id
was used, a universal indicator was used instead of
meth yl red indicator.
Concentration and Ammonium Sulphate
Fractionation of Toxoid Using Pellicon System
After detox ifi cat ion and specific toxici ty testing. th e
ne xt important aspec t was the selection of ultrafilratio n
membrane for co ncentratin g th e toxoid. The selection
of th e device was based on th e criteria of void-free
c hara cter istic s. integ rity testab le, lowe r bindin g to
pro tein s and hi g he r flux. Jt also offe red g reater
mechanical strength and offerrecl fl exibility in cleaning.
The selection of Biomax from Millipore of modified
Polycthylsu lphon e (PES) satisfied the cr it ical
req uirement of the cassette se lection . The se lecti on of
thin channel configuration enhan ced the flux aspect in
addition to th e benefit s offered by the basic Biomax
membrane. There was no problem of fouling due to cc li s
as it was primarily an appli cation in volving dissolved
molec ul es of proteins. The ba tc h size at this stage of
concentration of tetanus tox oid was in the ran ge of I040-
1060 L. The molecular wei ght of tetanus toxoid is arou nd
 776 J SC I IND RES VOL 60 OCTOBER 2001

I 50,000 daltons, which inc ludes the monomer, dime rs

and oligomers of vari ous sizes. The area of th e device Table 3-Parameters monitored during orc ration of rbnt
sca le system
Bi o max I OK NMWL cassettes was 50 ft 2 . The cassettes
Parameter Bat ch No .
were tested for integrity, befo re and after every batch
1467 1469 1479 1481 1488
and th e values were recorded .
Yi , L 1000 1000 1000 1000 1000
The diafiltration process was performed afte r th e Yr. L 26 25 25 20 30
second fracti on of ammonium sulph ate prec ipitati o n of Yr. L 1040 1020 1035 1040 1050
toxo id, a purifi cation step for sulph ate remova l. Thi s was Yd , L 60 60 60 60 80
carried out with norma l sa line and in vo lved I 0-12 wash Time, min 300 300 450 305 315
cycles. Flux , Lrnh 22.4 2 1.9 14.8 22.0 21.5
ATP, bar 1.2 1. 2 1.3 1.3 u
Results and Discussion Initi al, Lt/mL 80 90 110 II 0 100
Permeate, LflmL 90 80 100 11 0 90
The summ ary of the pil ot-scale trial s is gtven tn
Protei n recovery.
Table I . The trial s s howed bette r recovery and a lso
per cent 92.4 90.6 94.0 94.5 95.0
effective and economi c clarification of the te tanus toxin
fermentation due to good flux . The data were scaled up Vi - Initi al volume, Yr - Retentate volume, Yr - Permeate volume.
for process in g a I 000 L batc h and a lso processed w ith Yd- Di afilration volume ATP - Average trans-membrane pressure
the commercial-scale data on the oth e r unit operation. Flux- the permeate tlow is desc ribed in uni ts of liters/unit area/
The detail s of the syste m in pilot- a nd plant-scales are unit time Lf/mL- Unit to describe the antigen co ncentrati on
given in Tabl e 2.
Table !-Su mmary of Pilot scale tri als
Table 4--Comrarison of dead-end filtrat ion with tangential
tlow fi lt ration
Para meter Batch No. Parameter Dead -end Tangentia l
2 3 4 5 filtra tion tlow filtrat ion
Vi, L 100 100 100 100 100
Filtration time, h 6-7 5
Yr. L 5.1 2.0 2.0 3.0 2.0
Yp, L 102 102 105 106 106 Sanitization/Cleaning ti me, h 6 1.5 h
Yd , L 10 8 10 10 10 Total working time, h 12 h, minimum 6.5 h
Time, min 180 160 170 IRO ISO Product recovery, rer cent 88-90 95-98
Flux , Lmh 58 56 40 3R 3R Filtrate quality Needs polishing by Alread y 0.2p
ATP, bar 1.2 10. 1.3 1.3 1.3 further filtrati on filtered
Initial, U/mL 75 65 90 110 100
Permeate, LflmL 70 60 80 100 95
Protein recovery,
per cent 95.2 94. 1 93 .3 96.0 95 .0 For the performance of the pl ant sca le system based
o n paramete rs g iven in Tabl e 2, TFF exercises for
Vi - Init ia l volume. Yr - Rctcntate volume, Yp - Permeate volu me,
producti on size batc hes we re conducted and data for five
Yd- Diafilration volume ATP- Average trans-membrane pressure
Flux -the permeate flow is described in un its of liters/uni t area/ typica l batches a re g iven in Tabl e 3.
unit time Lt/mL- Unit to describe the anti ge n concentration
Th e te tanu s toxin c larifi cati o n by depth filtration
showed a recovery of 88-90 pe r cent in about 12-1 3 h,
Table 2-Details of system in Pilot and Plant Scale
including post ope ration saniti zat ion. T he TFF sys te m,
Para meter Pilot sca le Plant scale o n th e other hand , gave a better y ie ld of 95-98 pe r cent
a nd required only 6.5 h for the filtration, inc luding the
Batch vo lume, L 100 1000 post-operation saniti zat ion . Th e syste m' s performance ,
Filtration area per module, ft ~ 2 20 as compared to depth filtrati on is g iven in Table 4 .
Total Filtration area, 1'! 2 10 100 The TFF system offered foll owi ng add ition al bene fits:
Transmembrane pressure 1.0-1.5 1.0 - 1.5
of operat ion. bar Closed operati ons, no spill age and thu s mo re in
Protein recovery, per cent 95 - I00 95- 9R keep ing wi th GMP.
 RAV ETKAR eta!. : PROCESS I G OF TETAM US TO XI 777

Table 5-Details of processing of different production sca le batches using TFF syste m

Parameter Crude tox in lot No.

1467 1469 1471 1475 1477

Filtration date 29/03/97 09/04/97 19/04/97 I0/05/97 20/05/97

Volume, L 1000 1000 1000 1000 1000
Permeate, Lf/mL 80 80 90 100 90
Saline used, L 60 60 60 60 60
Filterate, L 1040 1020 1025 1020 1035
Toxidation completion date 28/04/97 09/05/97 19/05/97 09/06/97 19/06/97
Concentration date 19/05/97 3 1/05/97 09/06/97 01/07/97 11/07/97
Concentration volume, L 42 41 40 52 42
Concentrate, Lf/mL 1900 1900 2000 2000 2000
Concentrate recovery, per cent 96 95 87 100 90
Salting out date 20/05/97 02/06/97 11/06/97 03/07/97 13/07/97
Final filtration date 23/05/97 04/06/97 13/06/97 05/07/97 16/07/97
Final filt rate volume, L 19.7 20.0 20.0 20.0 19.8
Filtrate, Lf/mL 3200 3200 3300 4250 3500
Fractionation recovery, per cent 79 82 83 82 83
Purity, Lf/mg PN 1957 1852 1670 1782 1852
l
Total recovery, per cent 76 78 72 83 74

Crude toxin lot No.

1479 1481 1482 1484 1488

Filtration date 30/5/97 10/06/97 20/06/97 30/06/97 21/07/97

Volume, L 1000 1000 1000 1000 1000
Permeate, Lti'ml 100 100 100 80 90
Saline used, L 60 60 60 60 80
Filtered volume, L 1035 1040 1020 1030 1050
Toxidation completion da~ 29/06/97 I0/07/97 20/07/97 30/07/97 20/08/97
Concentrated on, date 2 1/07/97 0 1/08/97 II /08/97 23/08/97 I 0/09/97
Concentrate volume, L 47 50 52 37 43
Permeate in concentrate, Lf/mL 2000 1900 1900 1900 1800
Concentration recovery, per cent 91 91 97 85 82
Salting out date 23/07/97 03/08/97 13/08/97 25/08/97 14/09/97
Final filtration date 26/07/97 06/08/97 17/08/97 27/08/97 17/09/97
Final filtrate volume, L 20.0 20.0 20.0 20.0 20.0
Final filtrate , Lf/m L 3900 3750 4200 2750 3000
Fractionation recovery, per cent 83 76 85 78 78
Purity, Lti'mg PN 1800 1720 1929 1679 1542
l
778 J SCI INO RES VOL 60 OCTOBER 200 1
Minimal ex posure of the working personn el.
No rec urrin g cost of pad s, etc.
Savings on autocla vin g charges for sterili zati on
of filter press asse mbli es.
Sav in gs o n disp osa l ex pe ns es lik e
decontamination, and incinerat ion of used pads .
o problem of final sterili zatio n- filtrati on. as
0.2 Jl filtered output was used.
Validation of TFF System
The pre- and post-u sage integrit y tes tin g showed
repeatable use of membran e device and fulfillment of
an important GMP norm. For removal of formalin , the
system must be flu shed with I00 L of di sti lied water
whereby the concentration of formalin is reduced below
5 ppm.
Absence of Cl tetani colony on th e GVWP disc
confirmed that no organi sm was present in th e clarified
broth . The positive growth of the organism on M & M
agar also confirmed th at the medium could support the
growth of Cl tetani .
In th e case of removal of acetic acid from th e sys tem,
the test sample obtained after pass ing 50 L di still ed water
had colour simil ar to th at of with I 0 ppm acet ic acid
after add ition of meth yl red indicator. The colour chan ge
below thi s concentration was not di stin guishable. To
ensure th at no trace of the chemical remained, anoth er
50 L di still ed water was flush ed through the sys tem.
In the case of th e remo va l of sodium hypochl orite
from the system, the test sample obtained after pass ing
50 L di still ed water had colour similar to I 0 ppm sod ium
hypoc hl orite after addition of th e uni ve rsa l indi cator.
The colour change be low thi s concentrati on was not
distinguishable, and to furth er ensure that no trace of
th e chemical remained, anoth er 50 L di still ed water was
flush ed throu gh th e system.
Thu s, from GMP point of view th e sys tem was
co mpl etel y va lidated .
Concentration and Ammonium Sulphate
Fractionation of Toxoid Using Pellicon System
As desc rib ed above , afte r co mpl e ti o n of
detox ificati on, th e toxoid was co ncentrated, ammonium
sulphate fracti onated and fin all y steril e fil te red. The steps
involved , s uc h as c la rifi ca ti o n. co nce ntrati on,
diafiltration and ste ril e filtration , in th e production of
tetanus toxoid are summari zed in Tabl e 5.
The advantages of usin g a T FF sys tem ha ve bee n
di scussed above. The benefits of Pellicon sys tem used
for diafiltrati on are: (i) It reduces th e time required in
dialysis, (ii ) The system can be va lidated as per the GMP
requirement, and (iii ) There is minimum ex pos ure of
working perso nn el, as it is a closed system.
Acknowledgments
We are ex tremely thankful to Dr C S Poonawalla.
Pres ident and Dr J M Mehta , vice-president, Serum
Institute of India Research Foundati on for their kind help
durin g th e resea rch on thi s project.
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2 Levine, H.L. & Castillo, F.J , In 1/iotechnologr. qua/it\' as.wrance
and validation , Drug M anufac tcring Tech no logy Series. ed ited
by K E Avis. C M Wagner and V L Wu ( Inter Pharma Press Inc.
Bullalo Grove. Illinois) Vol. 4, 1999 , I 54.
3 WHO, Manualfor the flroduction & contm l of' vaccine: TetWIIIS
toxoid (World ll ea lth Org~nizationlBLG/ U DP/77.2 Rev. I